Process for preparing optically active 2-[6-(hydroxy-methyl)-1,3-dioxan-4-yl] acetic acid derivatives

ABSTRACT

The present invention is to provide a production technology by which an optically active 2-[6-(hydroxymethyl)-1,3-dioxan-4-yl]acetic acid derivative, which are of value as pharmaceutical intermediates, can be produced from inexpensive and readily available starting materials without using any extraordinary equipment such as an ultra-low-temperature reactor. The present invention is a production process of an optically active 2-[6-(hydroxymethyl)-1,3-dioxan-4-yl]acetic acid derivative which comprises reacting an enolate, prepared by permitting a base or a 0-valent metal to act on an acetic acid ester derivative with (S)-β-hydroxy-γ-butyrolactone at a temperature not lower than −30° C. to give a dihydroxyoxohexanoic acid derivative, treating the same with an acylating agent in the presence of a base to produce a dihydroxyoxohexanoic acid monoacyl derivative, reducing this compound with a microorganism to produce a trihydroxyhexanoic acid monoacyl derivative, treating this compound with an acetal-forming reagent in the presence of an acid catalyst to produce an acyloxy-methyldioxanylacetic acid derivative, and finally, subjecting this compound to solvolysis in the presence of a base.

TECHNICAL FIELD

The present invention relates to production process of pharmaceuticalintermediates, particularly optically active2-[6-(hydroxymethyl)-1,3-dioxan-4-yl]acetic acid derivatives of value asintermediates of HMG-COA reductase inhibitors.

BACKGROUND ART

The hitherto-known technology for producing2-[6-(hydroxymethyl)-1,3-dioxan-4-yl]acetic acid derivatives includesthe following processes.

(1) A process starting with 3-hydroxy-γ-butyrolactone to synthesize a3,5,6-trihydroxyhexanoic ester derivative via a 3,5-dihydroxyhexanoicester derivative (JP-A-04-173767).

(2) A process starting with 3,4-dihydroxybutyronitrile acetonide tosynthesize a 3,5,6-trihydroxyhexanoic ester derivative via a3,5-dihydroxyhexanoic ester derivative (JP-A-02-262537).

(3) A process starting with a 4-chloroacetoacetic acid ester tosynthesize a 3,5,6-trihydroxyhexanoic ester derivative throughbenzyloxylation, reduction, chain extension and like steps.(JP-A-06-65226).

(4) A process starting with a 4-chloro-3-hydroxybutyric ester tosynthesize a 3,5,6-trihydroxyhexanoic ester derivative through chainextension, reduction and like steps. (U.S. Pat. No. 5,278,313).

(5) A process starting with malic acid to synthesize a3,5,6-trihydroxyhexanoic ester derivative via a 2,4-dihydroxyadipic acidderivative (JP-A-04-69355).

However, these processes involve ultra-low temperature reactions around−80° C. in some stage or other of the respective production processes(1,2,4 and 5) or a high-pressure hydrogenation reaction requiring apressure of as high as 100 kg/cm² (3), thus invariably requiringextraordinary reaction equipment. Moreover, expensive starting materialsare used in some or other stages, so that none of the processes areefficient enough for industrial-scale production.

SUMMARY OF THE INVENTION

In the above state of the art, the object of the present invention is toprovide a production technology by which an optically active2-[6-(hydroxymethyl)-1,3-dioxan-4-yl]acetic acid derivative representedby the following formula (I), which are of value as pharmaceuticalintermediates, can be produced with ease and high efficiency frominexpensive starting materials without using any extraordinary equipmentsuch as an ultra-low-temperature reactor;

in the formula, R¹ represents a hydrogen, an alkyl group of 1 to 12carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl groupof 7 to 12 carbon atoms; R² and R³ each independently represents ahydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; and R andR³ may jointly form a ring.

Under the circumstances, the inventors of the present invention carriedout intensive investigations and, as a consequence, developed anexpedient technology for producing optically active2-[6-(hydroxymethyl)-1,3-dioxan-4-yl]acetic acid derivatives of thefollowing formula (I) from inexpensive, readily available startingmaterials without using any extraordinary equipment such as anultra-low-temperature reactor;

in the formula, R¹ represents a hydrogen, an alkyl group of 1 to 12carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl groupof 7 to 12 carbon atoms; R² and R³ each independently represents ahydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; and R² andR³ may jointly form a ring.

The present invention, therefore, is directed to a production process ofan optically active 2-[6-(hydroxymethyl)-1,3-dioxan-4-yl]acetic acidderivative represented by the general formula (I);

in the formula, R¹, R² and R³ are as defined above,

which comprises

(1) reacting an enolate prepared by permitting a base or a 0-valentmetal to act on an acetic acid ester derivative represented by thefollowing formula (II);

in the formula, R¹ is as defined above; and X¹ represents a hydrogen ora halogen atom,

with (S)-β-hydroxy-γ-butyrolactone represented by the following formula(III);

at a temperature not lower than −30° C. to produce a compoundrepresented by the following formula (IV);

in the formula, R¹ is as defined above,

(2) treating this compound with an acylating agent in the presence of abase to produce a compound represented by the following formula (V);

in the formula, R¹ is as defined above; and R⁴ represents a hydrogen, analkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbonatoms, or an aralkyl group of 7 to 12 carbon atoms,

(3) reducing this compound with a microorganism to produce a compoundrepresented by the following formula (VI);

in the formula, R¹ and R⁴ are as defined above,

(4) treating this compound with an acetal-forming reagent in thepresence of an acid catalyst to produce a compound represented by thefollowing formula (VII);

in the formula, R¹, R², R³, and R⁴ are as defined above,

and (5) subjecting this compound to solvolysis in the presence of abase.

The present invention is also directed to an isolation/purificationprocess

which comprises treating a compound contaminated with an impurity andrepresented by the following formula (V);

in the formula, R¹ represents a hydrogen, an alkyl group of 1 to 12carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl groupof 7 to 12 carbon atoms; and R⁴ represents a hydrogen, an alkyl group of1 and 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or anaralkyl group of 7 and 12 carbon atoms,

with an aliphatic hydrocarbon solvent to remove the impuritycontaminating the compound represented by the above formula (V) and

obtaining the compound represented by the above formula (V) in a crystalform.

Furthermore, the present invention is directed to a production processof a compound represented by the following formula (VI);

in the formula, R¹ and R⁴ are as defined above,

which comprises reducing a compound represented by the following formula(V);

in the formula, R¹ and R⁴ are as defined above,

with a microorganism.

In another aspect, the present invention is directed to a productionprocess of a compound represented by the following formula (VII);

in the formula, R¹, R², R³ and R⁴ are as defined above,

which comprises treating a compound represented by the following formula(VI);

in the formula, R¹ and R⁴ are as defined above,

with an acetal-forming reagent using an amine salt composed of an acidand an amine as a catalyst.

In still another aspect, the present invention is directed to anisolation/purification process

which comprises treating a compound represented by the following formula(VI);

in the formula, R¹ and R⁴ are as defined above,

with an acetal-forming reagent in the presence of an acid catalyst tothereby convert the same to a compound represented by the followingformula (VII);

in the formula, R¹, R², R³ and R⁴ are as defined above,

treating the compound contaminated with an impurity and represented bythe above formula (VII) with an aliphatic hydrocarbon solvent to removethe impurity contaminating the compound represented by the above formula(VII) and

obtaining the compound represented by the above formula (VII) in acrystal form.

DISCLOSURE OF INVENTION

The present invention is now described in detail.

As illustrated in the following reaction scheme, the present inventioncomprises five non-ultra-low temperature reaction steps (1) through (5).

The present invention is now described, step by step, in detail.

Step (1)

In this step, an enolate prepared by permitting a base or a 0-valentmetal to act on an acetic acid ester derivative represented by thefollowing formula (II);X¹CH₂CO₂R¹  (II)is reacted with (S)-β-hydroxy-γ-butyrolactone represented by thefollowing compound (III);

at a temperature not lower than −30° C. to produce a (5S)-configureddihydroxyoxohexanoic acid derivative represented by the followingformula (IV);

Generally when a reaction involving the enolate of an acetic acid esteror the like is carried out in a non-ultra-low temperature reaction over,for example, not lower than −30° C., the self-condensation of theenolate proceeds preferentially to markedly sacrifice the conversionrate of the objective reaction. However, it was found that according tothe following technique developed by the present inventors, theself-condensation of the acetic acid enolate can be minimized and theobjective reaction can be carried through with good product yield.

(S)-β-hydroxy-γ-butyrolactone, which is used in the step (1), can beproduced in a large scale by the known technology (e.g. SYNTHETICCOMMUNICATION, 1986, 16, 183.).

Referring to the acetic acid ester derivative for use in the step (1),R¹ represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, anaryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12carbon atoms, and more specifically, including a hydrogen, methyl,ethyl, i-propyl, tert-butyl, n-octyl, phenyl, naphthyl, p-methoxyphenyl,p-nitrobenzyl, and like groups. The preferred is a tert-butyl group. X¹represents a hydrogen or a halogen atom, and more specifically includinga hydrogen, a chlorine, a bromine, and an iodine, preferably a hydrogenand a bromine.

The level of use of the acetic acid ester derivative relative to(S)-β-hydroxy-γ-butyrolactone is 1 molar equivalent to 10 molarequivalents, preferably 1 molar equivalent to 5 molar equivalents.

In the step (1), a base or a 0-valent metal is permitted to act on theacetic acid ester derivative to prepare an enolate. Generally speaking,in preparing the enolate, a base is used when X¹ of the acetic acidester is a hydrogen; a 0-valent metal is used in preparing the enolatewhen X¹ is a halogen atom.

The base which is used in preparing the enolate includes, for example,lithium amides such as lithium amide, lithium diisopropylamide, lithiumdicyclohexylamide, lithium hexamethyldisilazide, etc.; magnesium amidessuch as chloromagnesium diisopropylamide, bromomagnesiumdiisopropylamide, iodomagnesium diisopropylamide, chloromagnesiumdicyclohexylamide, etc.; sodium amides such as sodium amide, sodiumdiisopropylamide, etc.; potassium amides such as potassium amide,potassium diisopropylamide, etc.; alkyllithiums such as methyllithium,n-butyllithium, phenyllithium, tert-butyllithium, etc.; Grignardreagents such as methylmagnesium bromide, phenylmagnesium chloride,iso-propylmagnesium chloride, tert-butylmagnesium chloride, etc.; metalalkoxides such as sodium methoxide, magnesium ethoxide, potassiumtert-butoxide, etc.; and metal hydrides such as lithium hydride, sodiumhydride, potassium hydride, calcium hydride, and so forth. The preferredare metal hydrides, magnesium amides, lithium amides and Grignardreagents. These bases can be used each independently or in combination.For example, a lithium amide or a metal hydride is effective when usedin combination with a magnesium-containing base such as a Grignardreagent or a magnesium amide. Moreover, said magnesium-containing basemay be prepared in situ from a base and a magnesium compound such asmagnesium chloride, magnesium bromide, or the like.

The magnesium amide is represented by the general formula (VIII);

in the above formula, R⁵ and R⁶ each independently represents an alkylgroup of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, anaralkyl group of 7 to 12 carbon atoms, or a silyl group. Specifically,there can be mentioned methyl, ethyl, i-propyl, tert-butyl, cyclohexyl,n-octyl, phenyl, naphthyl, p-methoxyphenyl, p-nitrobenzyl,trimethylsilyl, triethylsilyl, phenyldimethylsilyl, and like groups. Thepreferred is an isopropyl group. X² represents a halogen atom and ispreferably a chlorine, a bromine, or an iodine. The more preferred is achlorine.

It is to be understood that the magnesium amide can be prepared from aninexpensive, readily available secondary amine and a Grignard reagent bythe known technology (for example, JP-A-08-523420). As an alternative,it can be prepared from lithium amide and a magnesium halide by theknown technology (e.g. J. Org. Chem. 1991, 56, 5978-5980).

The Grignard reagent is represented by the following formula (IX);R⁷—Mg—X³  (IX)in the above formula, R⁷ represents an alkyl group of 1 to 12 carbonatoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7to 12 carbon atoms. Specifically, there can be mentioned methyl, ethyl,n-propyl, i-propyl, n-butyl, tert-butyl, n-octyl, phenyl, naphthyl,p-methoxyphenyl, p-nitrobenzyl and like groups. The preferred groupsinclude methyl, ethyl, i-propyl, n-butyl, tert-butyl and so forth. X³represents a halogen atom and preferably is a chlorine, a bromine, or aniodine. The more preferred is a chlorine.

The lithium amide is represented by the general formula (X);

in the above formula, R⁸ and R⁹ each independently represents an alkylgroup of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, anaralkyl group of 7 to 12 carbon atoms, or a silyl group. Specifically,there can be mentioned methyl, ethyl, i-propyl, tert-butyl, cyclohexyl,n-octyl, phenyl, naphthyl, p-methoxyphenyl, p-nitrobenzyl,trimethylsilyl, triethylsilyl, phenyldimethylsilyl and like groups. Thepreferred is an isopropyl group.

The level of use of the base in the step (1), relative to(S)-β-hydroxy-γ-butyrolactone, is 1 molar equivalent to 20 molarequivalents, preferably 2 molar equivalent to 8 molar equivalents.

The 0-valent metal usable in preparing the enolate of the step (1)includes zinc, magnesium, tin, etc., although zinc and magnesium arepreferred. The level of use of the 0-valent metal relative to(S)-β-hydroxy-γ-butyrolactone is 1 molar equivalent to 20 molarequivalents, preferably 2 molar equivalent to 8 molar equivalents.

The solvent usable in the step (1) includes, for example, aproticorganic solvents. As such organic solvents, there can be mentioned, forexample, hydrocarbon solvents such as benzene, toluene, n-hexane,cyclohexane, etc.;

ether solvents such as diethyl ether, tetrahydrofuran, 1,4-dioxane,methyl t-butyl ether, dimethoxyethane, ethylene glycol dimethyl ether,etc.; halogenated hydrocarbon solvents such as methylene chloride,chloroform, 1,1,1-trichloroethane, etc.; and aprotic polar solvents suchas dimethylformamide, N-methylpyrrolidone, hexamethylphosphorictriamide, and so forth. The above solvents may be used eachindependently or in a combination of two or more species. The preferred,among the above-mentioned solvents, are hydrocarbon solvents suchbenzene, toluene, n-hexane, cyclohexane, etc.; and ether solvents suchas diethyl ether, tetrahydrofuran, 1,4-dioxane, methyl t-butyl ether,dimethoxyethane, diethylene glycol dimethyl ether, and so forth.

The more preferred are polyether solvents such as dimethoxyethane,diethylene glycol dimethyl ether, and so forth.

These polyether solvents may be used each as a sole solvent or added, asan addendum, to another reaction solvent, at the level of about 1 molarequivalent to about 10 molar equivalents relative to(S)-β-hydroxy-γ-butyrolactone.

The reaction temperature to be used in the step (1) is preferably −30°C. to 100° C., more preferably −10° C. to 60° C.

In the step (1), the order of mixing the reactants may be random but itis preferred to treat (S)-β-hydroxyγ-butyrolactone with a base, morepreferably with a base and a magnesium compound in advance. Thepreferred base includes metal hydrides and lithium amides. The preferredmagnesium compound includes magnesium chloride, magnesium bromide,magnesium sulfate, and so forth. A magnesium-containing base may be usedso that it will double as the base and the magnesium compound. Themagnesium-containing base includes, for example, Grignard reagents suchas methylmagnesium bromide, iso-propylmagnesium chloride,phenylmagnesium chloride, tert-butylmagnesium chloride, etc. andmagnesium amides such as chloromagnesium diisopropylamide,bromomagnesium diisopropylamide, iodomagnesium diisopropylamide,chloromagnesium dicyclohexylamide and so forth. The preferred istert-butylmagnesium chloride.

The level of use of the base in this pretreatment, relative to(S)-β-hydroxy-γ-butyrolactone, is 0.01 molar equivalent through 3 molarequivalents, preferably 0.5 molar equivalent to 1.5 molar equivalents.

The level of use of the magnesium compound in the pretreatment, relativeto (S)-β-hydroxy-γ-butyrolactone, is 0.01 molar equivalent to 3 molarequivalents, preferably 0.5 molar equivalent to 1.5 molar equivalents.

The level of use of the magnesium-containing base in the pretreatment,relative to (S)-β-hydroxy-γ-butyrolactone, is 0.01 molar equivalent to 3molar equivalents, preferably 0.5 molar equivalent to 1.5 molarequivalents.

The pretreatment of (S)-β-hydroxy-γ-butyrolactone with the base may becarried out on a mixed solution of (S)-β-hydroxy-γ-butyrolactone and anacetic acid ester derivative. After the pretreatment, the reaction maybe conducted under dropwise addition of a base, such as a lithium amide,e.g. lithium amide, lithium diisopropylamide, lithium dicyclohexylamide,lithium hexamethyldisilazide or the like or a magnesium amide, e.g.diisopropylmagnesium chloride, diisopropylmagnesium bromide or the like,or a solution of the base.

The level of the base to be reacted after the pretreatment, relative to(S)-β-hydroxy-γ-butyrolactone, is 1 molar equivalent to 20 molarequivalents, preferably 2 molar equivalents to 8 molar equivalents.

Thus, the step (1) can be carried out with advantage by pretreating(S)-β-hydroxy-γ-butyrolactone with the base and the magnesium compoundin advance followed by permitting the base to act in the presence of theacetic acid ester derivative. As an alternative,(S)-β-hydroxy-γ-butyrolactone may be treated with the base in advanceand reacted with the enolate prepared by permitting a 0-valent metal toact on the acetic acid ester derivative.

The after-treatment following the step (1) may be an after-treatmentwhich is generally carried out for recovery of the product from areaction mixture. For example, the reaction mixture available oncompletion of the reaction is mixed with the common inorganic or organicsolvent, e.g. hydrochloric acid, sulfuric acid, nitric acid, acetic acidor citric acid followed by extraction with the common extractantsolvent, e.g. ethyl acetate, diethyl ether, methylene chloride, toluene,or hexane. From the extract thus obtained, the reaction solvent and theextractant solvent are removed by heating under reduced pressure or thelike procedure to give the objective compound. The objective compoundthus obtained may further be purified by the routine technique such ascrystallization, fractional distillation or column chromatography but itcan be transmitted directly to the next step without isolation.

Step (2)

In this step, the dihydroxyoxohexanoic acid derivative represented bythe following formula (IV);

as obtained in the step (1), is treated with an acylating agent in thepresence of a base to produce a dihydroxyoxohexanoic acid monoacylderivative represented by the following formula (V);

As the acylating agent usable in the step (2), any of a compoundrepresented by the following formula (XI);

and a compound represented by the following formula (XVI);

can be used. In the above formulas, R⁴ represents a hydrogen, an alkylgroup of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, oran aralkyl group of 7 to 12 carbon atoms. Specifically, there may bementioned a hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl,tertbutyl, n-octyl, phenyl, naphthyl, p-methoxyphenyl, p-nitrobenzyl,and like groups. The preferred are methyl, ethyl, i-propyl, tert-butyl,and phenyl groups, with a phenyl group being particularly preferred.

Q represents a leaving group. Specifically, there can be mentioned ahalogen atom, e.g. chlorine, bromine, iodine, etc.; an alkoxycarbonyloxygroup, e.g. methoxycarbonyloxy, ethoxycarbonyloxy,tert-butoxycarbonyloxy, etc.; a cyano group; an imidazolido group, andso forth. The preferred is a chlorine.

The level of use of the acylating agent relative to thedihydroxyoxohexanoic acid derivative is preferably 0.5 to 2 molarequivalents, more preferably 0.8 to 1.5 molar equivalents.

The base which can be used in the step (2) includes inorganic bases suchas sodium carbonate, potassium carbonate, sodium hydrogen carbonate,potassium hydrogen carbonate, sodium hydroxide, potassium hydroxide,calcium hydroxide, lithium hydroxide, barium hydroxide, magnesiumhydroxide, etc.; ammonia and amines such as triethylamine, pyridine,N-methylmorpholine, diisopropylethylamine, N,N-dimethylaminopyridine,etc., with triethylamine or pyridine being preferred. The level of useof the base relative to the dihydroxyoxohexanoic acid derivative ispreferably 1 to 10 molar equivalents, more preferably 1 to 3 molarequivalents.

The reaction solvent which can be used in the step (2) includeshydrocarbon solvents such as benzene, toluene, cyclohexane, etc.; ethersolvents such as diethyl ether, tetrahydrofuran, 1,4-dioxane, methyltert-butyl ether, dimethoxyethane, etc.; ester solvents such as ethylacetate, butyl acetate, etc.; ketone solvents such as acetone, methylethyl ketone, etc.; halogenated hydrocarbon solvents such as methylenechloride, chloroform, 1,1,1-trichloroethane, etc.; nitrogen-containingsolvents such as dimethylformamide, acetamide, formamide, acetonitrile,etc.; and aprotic polar solvents such as dimethyl sulfoxide,N-methylpyrrolidone, hexamethylphosphoric triamide, and so forth. Theabove-mentioned organic solvents may be used each independently or in acombination of two or more species. The preferred are toluene, ethylacetate, acetone, methylene chloride, methyl tert-butyl ether,tetrahydrofuran, dimethylformamide, acetonitrile, and so forth.

The reaction temperature in the step (2) is −30° C. to 80° C.,preferably −10° C. to 40° C.

The after-treatment following the step (2) may be an after-treatmentwhich is generally carried out for recovery of the product from thereaction mixture on completion of the reaction. For example, thereaction mixture available on completion of the reaction is added withwater and extraction procedure is carried out with the common extractantsolvent, such as ethyl acetate, diethyl ether, methylene chloride,toluene, hexane or the like. From the extract thus obtained, thereaction solvent and extractant solvent are removed by heating underreduced pressure or the like procedure to obtain the objective compound.

The objective compound thus obtained tends to contain various impuritiesoriginating from various decompositions and side reactions which takeplace in the course of production. Particularly, thedihydroxyoxohexanoic acid diacyl derivative of the following generalformula (XII);

(R¹ and R⁴ are as defined hereinbefore) tends to be by-produced as amajor impurity and in order that the objective compound of high qualitygrade may be isolated, such impurities must be somehow removed.Generally, however, an impurity structurally resembling the objectivecompound (structural analog) is not easy to remove, and in order thatsuch impurities may be removed to give the objective compound of highpurity grade, a good protocol for purification and isolation isrequired. The inventors of the present invention found that saidimpurities can be efficiently removed by carrying out a crystallizationprocedure under the conditions described below.

The crystallization solvent for use in the present invention ispreferably an aliphatic hydrocarbon solvent. Specifically, aliphatichydrocarbons containing 5-20 carbon atoms, such as pentane, petroleumether, neopentane, hexane, cyclohexane, methylcyclohexane, heptane,cycloheptane, octane, isooctane, nonane, decane, undecane, dodecane,etc. can be mentioned. Among these, pentane, hexane, methylcyclohexane,heptane, octane, and isooctane are preferred. These may be used eachindependently or in a combination of two or more species.

Particularly in terms of the ease of removal of the solvent from wetcrystals by desiccation or the recovery and reuse of the solvent(distillative recovery), the use of a solvent having a comparatively lowboiling point is preferred. As such solvents, solvents having a boilingpoint of not higher than about 100° C. at atmospheric or subatmosphericpressure can generally be mentioned. More particularly, aliphatichydrocarbon solvents of 5-8 carbon atoms, such as pentane, hexane,methylcyclohexane, heptane, octane and isooctane, etc. can be mentioned,and when the cost of the solvent, ease of handling, and other factorsare globally taken into consideration, hexane and methylcyclohexane areparticularly preferred.

The use of the above aliphatic hydrocarbon solvent provides for thestabilization and assurance of a high yield of the objective compound aswell as a high degree of purification, that is to say effective removalof various impurities, particularly said compound (XII). The level ofuse of said aliphatic hydrocarbon solvent is preferably such that, atcompletion of the procedure for crystallization of said compound (V),the fluidity of the obtained product can be retained, and may, forexample, be about 5 to 20 parts by weight, or even more in some cases,relative to said compound (V).

For the crystallization of said compound (V) in the present invention,crystallization by cooling, crystallization by concentration, and othermethods for crystallization can be used each independently or incombination. The crystallization by concentration, mentioned above, maybe a crystallization procedure in which a solution composed of a solventother than said aliphatic hydrocarbon solvent is converted to a solutioncomposed of said aliphatic hydrocarbon solvent. Moreover, seed crystalsmay be added in this crystallization procedure.

In the present invention, for the purpose of improving at least one of asolubility, yield, treatment concentration, purification effect(efficiency of impurity removal), and physical properties of obtainablecrystals of the above compound (V), an auxiliary solvent can be used inaddition to said aliphatic hydrocarbon solvent in conducting thecrystallization procedure. The above auxiliary solvent may be added tosaid aliphatic hydrocarbon solvent as necessary or the compound (V) maybe dissolved in the auxiliary solvent in advance and the solution addedto said aliphatic hydrocarbon solvent.

The auxiliary solvent mentioned above is not particularly restricted butincludes, for example, acetone, methyl ethyl ketone, tetrahydrofuran,methyl tert-butyl ether, ethyl acetate, isopropyl acetate, tert-butylacetate, ethanol, isopropanol, toluene, benzene, xylene, chlorobenzene,methylene chloride, chloroform, and 1,2-dichloroethane, etc. These maybe used each independently or in a combination of two or more species.Among these, ethyl acetate, toluene, methyl tert-butyl ether, methylenechloride, etc. can contribute to increased solubility and improvedtreatment parameters such as treatment concentration and purificationeffect.

The auxiliary solvent mentioned above expresses its effect moreprominently when used in a suitable amount combined with said aliphatichydrocarbon solvent, which suitable amount being established accordingto the characteristics of the auxiliary solvent in relation to thedesired effect and other factors. The optimal level of use of saidauxiliary solvent can be found by simple experimentation. From thestandpoint of yield and purification effect, the level of use of theabove auxiliary solvent is preferably such that the weight ratio of saidauxiliary solvent and said aliphatic hydrocarbon solvent (auxiliarysolvent/aliphatic hydrocarbon solvent) is not greater than 1 atcompletion of the procedure for crystallization of said compound (V).The more preferred level is such that e ratio id 0.5 or less.

The purification/isolation process according to the present inventioncan be carried out in the neighborhood of room temperature. Wherenecessary, warming or cooling can be carried out, for example at atemperature not over about 60° C., usually at −30° C. to 50° C.

The above compound (V) thus obtained is separated by a solid-liquidseparation technique, optionally further followed by cake washing anddrying. The solid-liquid separation technique is not particularlyrestricted but includes, for example, filtration under pressure, suctionfiltration, centrifugation, and so forth. The above drying is preferablycarried out under reduced pressure (drying in vacuo) at a temperaturenot exceeding about 60° C. in order to avoid pyrolysis or fusion, forinstance.

Step (3)

In this step, the (5S)-configured dihydroxyoxohexanoic acid monoacylderivative represented by the following formula (V);

as obtained in said step (2) is reduced using a microorganism to give a(3R,5S)-configured trihydroxyhexanoic acid monoacyl derivativerepresented by the following formula (VI);

Generally for the highly stereoselective reduction of the carbonyl groupof a dihydroxyoxohexanoic acid monoacyl derivative such as the abovecompound, the reduction process using a hydride series reducing agent,such as sodium borohydride, in the presence of an alkylborane at anultra-low temperature is employed (e.g. JP-A-02-262537).

For the purpose of reducing a dihydroxyoxohexanoic acid monoacylderivative stereoselectively under non-ultra-low temperature conditionsat low cost, the inventors of the present invention developed areduction method using a microorganism.

The microorganism capable of reducing dihydroxyoxohexanoic acid monoacylderivatives to the corresponding trihydroxyhexanoic acid monoacylderivatives, which is to be used in the step (3), can be found by themethods described below. Taking a yeast as an example, there can beused, for example, the method which comprises charging a large test tubewith 5 ml of Medium A (pH 7.0) of the composition: glucose 3%, yeastextract 0.3%, potassium dihydrogen phosphate 0.7%, diammonium hydrogenphosphate 1.3%, magnesium sulfate.7H₂O 0.08%, zinc sulfate.7H₂O 0.007%,iron sulfate.7H₂O 0.009%, copper sulfate.5H₂O 0.0005%, manganesesulfate.4H₂O 0.001%, and sodium chloride 0.01%, sterilizing the medium,inoculating a yeast, carrying out a shake culture at 27° C. for 2 to 3days, harvesting the grown cells by centrifugation, suspending the cellsin 0.5 ml of phosphate buffer containing 0.01 to 1% of(5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic acid tert-butyl ester and 8%of glucose, and shaking the suspension in a large test tube at 27° C.for 1 to 3 days. For the identification of the objective reducingability, the reaction mixture after shake culture is extracted withethyl acetate and the organic phase is analyzed by high performanceliquid chromatography [column: Dovelosil ODS-HG-3 (4.6 mm×150 mm)(product of Nomura Chemical), eluent: 0.1% trifluoroaceticacid/acetonitrile=6/4, flow rate: 0.8 ml/min, detection: 210 nm, columntemperature: room temperature, elution time:(3S,5S)-6-benzoyloxy-3,5-dihydroxyhexanoic acid tert-butyl ester; 10.1min, (3R,5S)-6-benzoyloxy-3,5-dihydroxyhexanoic acid tert-butyl ester;11.0 min, and (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic acid tert-butylester; 16.7 min; it is to be understood that these conditions are forillustrative purposes only]. When the candidate microorganism is abacterial strain, Medium B (pH 7.0) of the composition: glycerol 1.5%,Proextract 1.0%, and yeast extract 0.5%, for instance, is employed, forinstance. In the case of a fungal strain, Medium C (pH 6.0) of thecomposition: glucose 5% and corn steep liquor 5% is employed, forinstance. In the case of an actinomycete, Medium D (pH 7.2) of thecomposition: Difco-tryptic soy broth 3% and soluble starch 1% isemployed. Using these media, the respective strains of microorganismsare cultured, and the microorganisms having the objective ability areselected by the same procedure as described above.

The microorganism which can be used in the practice of the presentinvention includes microorganisms belonging to the genera: Ashbya,Botryoascus, Brettanomyces, Candida, Citeromyces, Clavispora,Cryptococcus, Debaryomyces, Dekkera, Dipodascus, Galactomyces,Geotrichum, Hanseniaspora, Hansenula, Hormoascus, Hyphopichia,Issatchenkia, Kluyveromyces, Komagataella, Lipomyces, Metschnikowia,Nakazawaea, Ogataea, Pachysolen, Pichia, Rhodotorula, Rhodsporidium,Saccharomyces, Saccharomycodes, Saccharomycopsis, Saturnospora,Schizoblastosporion, Schizosaccharomyces, Schwanniomyces, Sporidiobolus,Sporobolomyces, Torulaspora, Torulopsis, Trichosporon, Trigonopsis,Willopsis, Yamadazyma, Zygosaccharomyces, Acidiphilium, Aerobacter,Alcaligenes, Arthrobacter, Aureobacterium, Bacillus, Brevibacterium,Buttiauxella, Cedecea, Cellulomonas, Citrobacter, Clostridium,Comamonas, Corynebacterium, Enterobacter, Erwinia, Escherichia,Flavobacterium, Klebsiella, Luteococcus, Microbacterium, Micrococcus,Ochrobactrum, Proteus, Providencia, Pseudomonas, Rhodococcus, Sarcina,Serratia, Sphingobacterium, Tsukamurella, Absidia, Acremonium, Aegerita,Agrocybe, Amylostereum, Aspergillus, Bvssochlamys, Chaetomidium,Chaetosartorya, Cladosporium, Coprinus, Crinipellis, Endophragmia,Flavolus, Fomitopsis, Fusarium, Ganoderma, Glomerella, Laetiporus,Lentinus, Lenzites, Macrophoma, Monascus, Mortierella, Paecilomyces,Penicillium, Phialophora, Pholiota, Pleurotus, Scopulariopsis,Sehizophyllum, Sporotrichum, Zygorhynchus, Microtetraspora, andStreptomyces. More particularly, the usable are, for example, Ashbyagossypii IFO 0560, Botryoascus synnaedendrus IFO 1604, Brettanomycescustersianus IFO 1585, Candida arborea IAM 4147, Candida catenulata IFO0745, Candida fennica CBS 6028, Candida galacta IFO 10031, Candidahaemulonii IFO 10001, Candida magnoliae IFO 0705, Candida musae IFO1582, Candida nitratophila IFO 10004, Candida parapsilosis IFO 0585,Candida pararugosa IFO 0966, Candida stellata IFO 0701, Citeromycesmatritensis IFO 0651, Clavispora lusitaniae IFO 1019, Cryptococcuslaurentii IFO 0609, Debaryomyces carsonii IFO 0795, Debaryomyceshansenii var. fabryi IFO 0794, Debaryomyces hansenii var. hansenii IFO0032, Debaryomyces hansenii var. hansenii IFO 0047, Debaryomyceshansenii var. hansenii IFO 0018, Debaryomyces kloeckeri, Debaryomycesmarama IFO 0668, Debaryomyces pseudopolymorphus IFO 1026, Debaryomycesrobertsiae IFO 1277, Debaryomyces sp. IFO 0025, Dekkera anomala IFO0627, Dipodascus armillariae IFO 0102, Dipodascus ovetensis IFO 1201,Dipodascus tetrasperma CBS 765.70, Galactomyces reessii CBS 179.60,Geotrichum candidum CBS 187.67, Geotrichum fermentans IFO 1199,Geotrichum fragrans CBS 164.32, Geotrichum loubieri CBS 252.61,Hanseniaspora guilliermondii IAM 4972, Hansenula methanolosa, Hansenulapolymorpha DL1 AKU4752, Hormoascus philentomus IFO 1847, Hormoascusplatypodis IFO 1471, Hyphopichia burtonii IFO 0844, Issatchenkiaorientalis IFO 1279, Issatchenkia terricola IFO 0933, Kluyveromyceslactis IFO 1012, Kluyveromyces marxianus IFO 0541, Kluyveromycesmarxianus IFO 0288, Kluyveromyces polysporus IFO 0996, Kluyveromycesthermotolerans IFO 0662, Komagataella pastoris IFO 1013, Lipomycesstarkeyi IFO 0678, Metschnikowia bicuspidata IFO 1408, Metschnikowiapulcherrima IFO 0561, Nakazawaea holstii IFO 0980, Ogataea minuta var.minuta IFO 0975, Ogataea pini IFO 1342, Ogataea polymorpha IFO 0799,Ogataea polymorpha IFO 1475, Ogataea wickerhamii IFO 1706, Pachysolentannophilus IFO 1007, Pichia canadensis IFO 0976, Pichia farinose IAM4369, Pichia jandinii IFO 0987, Pichia saitoi IAM 4945, Pichia toletanaIFO 0950, Pichia triangularis IFO 0836, Pichia wickerhamii IFO 1278,Rhodotorula graminis IFO 0190, Rhodotorula minuta IFO 0387, Rhodotorulaminuta IFO 0715, Rhodsporidium diobovatum IFO 0688, Rhodsporidiumtoruloides IFO 0413, Saccharomyces bayanus IFO 0251, Saccharomycespastorianus IFO 1265, Saccharomyces pastorianus ATCC 9080, Saccharomycesrosei IFO 0252, Saccharomyces sake, Saccharomyces steineri IAM 4608,Saccaromyces unisporus IFO 0215, Saccharomycodes ludwigii IFO 0339,Saccharomycopsis capsularis IFO 0672, Saccharomycopsis malanga IFO 1710,Saturnospora dispora IFO 0035, Schizoblastosporion kobayasii IFO 1644,Schizosaccharomyces pombe IFO 0347, Schizosaccharomyces pombe IFO 0362,Schwanniomyces occidentalis var. occidentalis IFO 1840, Sporidiobolusjohnsonii IFO 6903, Sporobolomyces pararoseus IFO 0471, Sporobolomycessalmonicolor IFO 1038, Torulaspora delbrueckii IFO 0381, Torulopsismethanolevescens, Torulopsis osboenis IFO 0646, Torulopsis sp.,Torulopsis uvae IFO 0649, Trichosporon pullulans, Trichosporon sp.Trigonopsis variabilis IFO 0671, Willopsis saturnus var. mrakii IFO0895, Willopsis saturnus var. saturnus IFO 0992, Yamadazyma farinosa IFO0459, Yamadazyma farinosa IFO 0602, Yamadazyma haplophila IFO 0947,Zygosaccharomyces naniwensis IFO 0524, Zygosaccharomyces sp. IFO 0522,Acidiphilium cryptum IFO 14242, Aerobacter cloacae IAM 1221, Alcaligenesxylosoxidans IFO 13495, Alcaligenes xylosoxidans subsp. denitrificansIFO 12669, Alcaligenes xylosoxidans subsp. denitrificans ATCC 15173,Arthrobacter globiformis ATCC 8010, Arthrobacter protophormiae IFO12128, Aureobacterium esteraromaticum IFO 3752, Bacillus badius IAM11059, Bacillus sphaericus IFO 3525, Brevibacterium ammomiagenes IFO12071, Buttiauxella agrestis JCM 1090, Cedecea davisiae JCM 1685,Cellulomonas sp. JCM 2471, Cellulomonas turbata IFO 15015, Citrobacterfreundii IFO 12681, Clostridium cylindrosporum IFO 13695, Comamonastestosteroni IFO 12047, Corynebacterium acectoacidophilum ATCC 21476,Corynebacterium ammoniagenes IFO 12072, Corynebacterium glutamicum ATCC21269, Corynebacterium glutamicus ATCC 13287, Enterobacter aerogenes IFO13534, Enterobacter cloacae IFO 12935, Erwinia carotovora subsp.carotovora IFO 3830, Escherichia coli IFO 12734, Flavobacteriumflavesceus, Klebsiella planticola IFO 3317, Luteococcus japonicus IFO12422, Microbacterium arborescens IFO 3750, Micrococcus flavus,Micrococcus luteus IFO 13867, Ochrobactrum sp. IFO 12950, Proteusinconstans IFO 12931, Proteus mirabilis IFO 3849, Proteus rettgeri IFO1350, Proteus vulgaris IFO 3167, Providencia stuartii IFO 12930,Pseudomonas aeruginosa IAM 1007, Pseudomonas putida IFO 14164,Pseudomonas stutzeri IFO 13596, Rhodococcus equi JCM 1313, Sarcinalutea, Serratia plymuthicum IFO 3055, Serratia proteamaculans subsp.proteamaculans IFO 12979, Sphingobacterium spiritivorum JCM 1277,Tsukamurella paurometabolum IFO 12160, Absidia orchidis HUT 1036,Acremonium bacillisporum IFO 9387, Aegerita candida IFO 6988, Agrocybecylindracea IFO 30299, Amylostereum areolatum IFO 9221, Aspergillusparasiticus IFO 4403, Aspergillus phoenicis IFO 6670, Byssochlamys fulvaIFO 6307, Chaetomidium fimeti IFO 30419, Chaetosartorya stromatoides IFO9652, Cladosporium resinae F. avellaneum IFO 6367, Coprinus cinereusTD-822, Coprinus lagopus IFO 9533, Coprinus sp., Crinipellis stipitariaIFO 30259, Endophragmia alternata IFO 30204, Flavolus arcularius,Fomitopsis pubertatis, Fusarium merismoides IFO 30040, Ganoderma lucidumIFO 31863, Glomerella cingulata IFO 5257, Laetiporus sulphureus,Lentinus lepideus, Lenzites betulina IFO 8715, Macrophoma commelinae IFO9569, Monascus purpureus IFO 5965, Mortierella isabellina IFO 7829,Paecilomyces varioti HUT 4028, Penicillium chermesinum IFO 5800,Penicillium chrysogenum IFO 4640, Penicillium expansum IFO 5854,Penicillium lilacinium IFO 31914, Phialophora fastigiata IFO 6850,Pholiota aurivella IFO 30265, Pholiota limonella IFO 31868, Pleurotusdryinus, Pleurotus ostreatus, Pleurotus porrigens, Scopulariopsisbrevicaulis IFO 4843, Sehizophyllum commune IFO 6503, Sehizophyllumcommune IFO 6504, Sporotrichum aurantiacum IFO 9381, Zygorhynchusmoelleri HUT 1305, Microtetraspora roseoviolacea IFO 14098, Streptomycesachromogenes subsp. rubradiris IFO 14000, Streptomyces sp. andStreptomyces aureus NIHJ 122.

These microorganisms can be obtained generally from stock cultures whichare readily available at no cost or at cost. These may also be isolatedfrom the natural kingdom. In this connection, these microorganisms mayoptionally be subjected to induced mutagenesis to obtain strains havingcharacteristics more beneficial to the intended reaction. Furthermore,the strains derived from these microorganisms by genetic engineering orbiotechnological processes, such as recombinant DNA technology or cellfusion may also be employed. As an example of such microorganism, therecan be mentioned Escherichia coli HB101 (pNTCRG) FERM BP-6898(PCT/JP00/08321) harboring the reductase gene derived from Candidamagnoliae IFO 0705.

For the cultivation of these microorganisms, any nutrient sources thatthese microorganisms are generally able to utilize can be employed. Forexample, as carbon sources, saccharides such as glucose, sucrose,maltose, etc.; organic acids such as lactic acid, acetic acid, citricacid, propionic acid, etc.; alcohols such as ethanol, glycerol, etc.;hydrocarbons inclusive of paraffin; oils such as soybean oil, rapeseedoil, etc.; and mixtures thereof, and as nitrogen sources, ammoniumsulfate, ammonium phosphate, urea, yeast extract, meat extract, peptone,corn steep liquor, etc. can be admixed. Furthermore, inorganic salts,vitamins, and other nutrients can also be properly admixed.

The above microorganisms can be cultured under the conventionalconditions. For example, they are cultured aerobically under the pH of4.0 to 9.5 in the temperature range of 20° C. to 45° C. for 10 to 96hours. To permit a microorganism to act on a dihydroxyoxohexanoic acidmonoacyl derivative, usually a culture of the above microorganism can beused as such for the reaction but a concentrate of the culture broth canlikewise be employed. Moreover, in cases where some ingredients in theculture broth might adversely affect the reaction, it is preferred touse the cells or processed cells as obtainable by centrifugation and/orother treatments on the culture broth.

The processed cells mentioned above are not particularly restricted butinclude, for example, dried cells obtainable by dehydration with acetoneor diphosphorus pentoxide or by drying with a desiccator or a fan,surfactanttreated cells, an enzymatic digest of cells, immobilizedcells, or a cell-free extract obtainable by disruption of cells.Furthermore, from the culture broth, an enzyme catalyzing asymmetricreduction may be purified and used.

In conducting the reduction reaction, the substrate dihydroxyoxohexanoicacid monoacyl derivative can be added all at once in the initial stageof the reaction or serially in portions with the progress of reaction.

The temperature during the reaction is generally 10 to 60° C.,preferably 20 to 40° C., and the pH during the reaction is 2.5 to 9,preferably 5 to 9.

The concentration of the microorganism in the reaction mixture can beproperly selected according to its ability to reduce the substrate. Theconcentration of the substrate in the reaction mixture is preferably0.01 to 50% (w/v), more preferably 0.1 to 30%.

The reaction is usually carried out under shaking or stirring withaeration. The reaction time is properly selected according to theconcentration of the substrate, the concentration of the microorganism,and other reaction conditions. Generally, the various conditions arepreferably selected so as to insure that the reaction may be completedin 2 to 168 hours.

To promote the reduction reaction, it is preferred to add an energysource, such as glucose, ethanol, and/or the like, in a proportion of 1to 30% to the reaction mixture, for it will lead to a more satisfactoryresult. The reaction can also be accelerated by adding a coenzyme, suchas reduced nicotinamide-adenine dinucleotide (NADH) or reducednicotinamide-adenine dinucleotide phosphate (NADPH), which is generallyconsidered to be necessary for reduction reactions by biologicaltechniques. More particularly, these may be directly added to thereaction mixture or a reaction system producing NADH or NADPH may beadded together with the oxidized coenzyme to the reaction mixture. Forexample, the reaction system which reduces NAD to NADH as a formatedehydrogenase produces carbon dioxide and water from formic acid or thereaction system which reduces NAD or NADP to NADH or NADPH respectively,as a glucose dehydrogenase produces gluconolactone from glucose can beexploited. Furthermore, it is also effective to add a surfactant such asTriton (product of Nakalai Tesque), Span (product of Kanto Chemical) orTween (product of Nakalai Tesque) to the reaction mixture. Moreover, forthe purpose of avoiding inhibition of the reaction by the substrateand/or alcohol, which is the byproduct of the reduction reaction, awater-insoluble organic solvent such as ethyl acetate, butyl acetate,isopropyl ether, toluene or the like may be added to the reactionmixture. For the purpose of enhancing the solubility of the substrate, awater-soluble organic solvent, such as methanol, ethanol, acetone,tetrahydrofuran, dimethyl sulfoxide, or the like, may also be added.

The trihydroxyhexanoic acid monoacyl derivative produced by thereduction reaction can be recovered, directly from the reaction mixtureor after removal of the cells and others, extraction with a solvent,such as ethyl acetate, toluene, or the like, and subsequent removal ofthe solvent. Moreover, the trihydroxyhexanoic acid monoacyl derivativecan be obtained in a highly pure grade by a purification procedure suchas recrystallization, silica gel column chromatography or the like.

Step (4)

In this step, the (3R,5S)-configured trihydroxyhexanoic acid monoacylderivative represented by the following formula (VI);

as obtained in the step (3), is treated with an acetal-forming reagentin the presence of an acid catalyst to produce a (4R,6S)-configuredacyloxymethyldioxanylacetic acid derivative represented by the followingformula (VII);

In the step (4), the acetal-forming reagent which can be used includes,for example, a ketone, an aldehyde, an alkoxyalkane, and analkoxyalkene. As specific examples of said ketone, aldehyde,alkoxyalkane and alkoxyalkene, there can be mentioned, for example,acetone, cyclohexanone, formaldehyde, benzaldehyde, dimethoxymethane,2,2-dimethoxypropane, 2-methoxypropene, and 1,1-dimethoxycyclohexane,etc. The preferred are acetone, 2-methoxypropene, 2,2-dimethoxypropane,etc. and more preferred is 2,2-dimethoxypropane. The level of use of theacetal-forming reagent relative to the trihydroxyhexanoic acid monoacylderivative is preferably 1 to 10 molar equivalents, more preferably 1 to5 molar equivalents. For promoting the reaction, the acetal-formingreagent can be used as the reaction solvent as well.

The acid catalyst which can be used in the step (4) includes Lewis acidsor Brönsted acids. As said Lewis acids and Brönsted acids, there can bementioned, for example, Lewis acids such as aluminum trichloride, borontrifluoride, zinc dichloride, tin tetrachloride, etc.; carboxylic acidssuch as oxalic acid, formic acid, acetic acid, benzoic acid,trifluoroacetic acid, etc.; sulfonic acids such as methanesulfonic acid,benzensulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, etc.;and inorganic acids such as hydrogen chloride, hydrogen bromide,sulfuric acid, nitric acid, boric acid, etc.

The level of use of the acid catalyst in the step (4), relative to thetrihydroxyhexanoic acid monoacyl derivative, is preferably 0.001 to 0.5molar equivalent, more preferably 0.005 to 0.1 molar equivalent.

The acetal-forming reaction using said acid catalyst is a side reactioninvolving the intramolecular hydroxyl groups, and it has heretofore beendifficult to let the desired reaction proceed with good efficiency.Regarding side reactions, a cyclization reaction involving theintramolecular hydroxyl groups and the ester group, for instance,produces an acyloxymethylhydroxylacetone represented by the followingformula (XV);

(in the formula, R⁴ is as defined hereinbefore) as a byproduct. When analkoxyalkane or alkoxyalkene is used as the acetal-forming reagent, thebyproduct alcohol reacts with the above compound (XV) to give an analogcompound (having different ester group) of saidacyloxymethyldioxalylacetic acid derivative represented by the followingformula (XIII);

(in the formula, R², R³ and R⁴ are as defined hereinbefore; and R¹⁰represents a lower alkyl group (preferably containing 1 to 4 carbonatoms) which is different from R¹) as a byproduct. When2,2-dimethoxypropane, for instance, is used as the acetal-formingreagent, the byproduct methanol takes part in the reaction to give anacyloxymethyldioxalylacetic acid methyl ester of the above formula(XIII) in which R¹⁰ represents a methyl group as a byproduct.

By-production of these impurities detract from the yield and quality ofthe objective product compound, so that when an acetal-forming reactionis to be conducted using said acid catalyst, the reaction conditionssuch as reaction temperature, reaction time, amounts of reagents, etc.are necessary to be critically selected and controlled.

The inventors of the present invention developed a method of conductingthe acetal-forming reaction in the presence of an amine salt composed ofan acid and an amine as a catalyst, whereby the formation of saidimpurities (XIII) and (XV) and several other trace byproduct impurities,which have not been structurally established, can be suppressed to aminimum without detracting from the yield. As the acid, any of the acidsmentioned hereinbefore can be employed, and the preferred includehydrogen chloride, hydrogen bromide, sulfuric acid, trifluoroaceticacid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonicacid. The level of use of the acid relative to the trihydroxyhexanoicacid monoacyl derivative is preferably 0.001 to 0.5 molar equivalent,more preferably 0.005 to 0.1 molar equivalent.

The amine includes ammonia; primary amines such as methylamine,ethylamine, butylamine, aniline, etc.; secondary amines such asdiethylamine, diisopropylamine, diphenylamine, piperidine, morpholine,etc.; and tertiary amines such as triethylamine, tributylamine,diisopropylethylamine, N-methylmorpholine, N-methylpiperidine, pyridine,2-methylpyridine, 3-methylpyridine, imidazole,N,N-dimethylaminopyridine, 1,7-diazabicyclo[5,4,0]-undec-7-ene, and soforth. The preferred are tertiary amines, with triethylamine,N-methylmorpholine, diisopropylethylamine, pyridine, 2-methylpyridine,3-methylpyridine or imidazole being more preferred. The level of use ofthe amine relative to the acid is preferably 1 to 10 molar equivalents,more preferably 1 to 3 molar equivalents.

This reaction may be carried out using an amine salt, as a catalyst,prepared from an acid and an amine and isolated in advance. The aminesalt includes, for example, pyridinium hydrochloride, pyridiniumhydrobromide, pyridinium sulfate, pyridinium trifluoroacetate,pyridinium methanesulfonate, pyridinium p-toluenesulfonate,triethylammonium hydrochloride, triethylammonium sulfate,3-methylpyridinium p-toluenesulfonate, N-methylmorpholinep-toluenesulfonate salt, N,N-dimethylaminopyridinium benzenesulfonate,diisopropylammonium hydrochloride, ammonium hydrochloride, ammoniumsulfate, ammonium nitrate, and ammonium methyl p-toluenesulfonate. Thepreferred is pyridinium p-toluenesulfonate or triethylammoniump-toluenesulfonate. The level of use of the amine salt relative to thetrihydroxyhexanoic acid monoacyl derivative is preferably 0.001 to 0.5molar equivalent, more preferably 0.005 to 0.1 molar equivalent.

For conducting the reaction according to the step (4), various organicsolvents can be used as the reaction solvent. As such organic solvents,there can be mentioned, for example, hydrocarbon solvents such asbenzene, toluene, cyclohexane, etc.; ether solvents such as diethylether, tetrahydrofuran, 1,4-dioxane, methyl t-butyl ether,dimethoxyethane, etc.; ester solvents such as ethyl acetate, butylacetate, etc.; ketone solvents such as acetone, methyl ethyl ketone,etc.; halogenated hydrocarbon solvents such as methylene chloride,chloroform, 1,1,1-trichloroethane, etc.; nitrogen-containing solventssuch as N,N-dimethylformamide, acetamide, formamide, acetonitrile, etc.;and aprotic polar solvents such as dimethyl sulfoxide,N-methylpyrrolidone, hexamethylphosphoric triamide, etc. These organicsolvents can be used each independently or two or more of them may beused in combination. The preferred are toluene, acetone, ethyl acetate,methylene chloride, tetrahydrofuran, methyl tert-butyl ether,dimethylformamide, and acetonitrile, with acetone being more preferred.

The reaction temperature in the step (4) is −20° C. to 100° C.,preferably OC to 50° C.

The after-treatment following the step (4) may be carried out by theroutine after-treatment for recovery of the product from a reactionmixture. For example, water is added to the reaction mixture aftercompletion of the reaction and the extraction is carried out with anordinary extractant solvent, for example, ethyl acetate, diethyl ether,methylene chloride, toluene, or hexane. From the extract thus obtained,the reaction solvent and extractant solvent are removed by heating underreduced pressure or the like procedure to give the objective compound.

The objective compound thus obtained tends to contain various impuritiesoriginating from various decompositions and side reactions which takeplace in the course of production. Particularly, it tends to contain atleast one member of the group consisting of an acyloxyhydroxylactonerepresented by the following general formula (XV);

(in the formula, R⁴ is as defined above), an analog compound (havingdifferent ester group) of an acyloxymethyldioxalylacetic acid derivativerepresented by the following general formula (XIII);

(in the formula, R², R³ and R⁴ are respectively as defined above; R¹⁰represents a lower alkyl group which is different from R¹), adiastereomer represented by the following formula (XIV);

(in the formula, R¹, R², R³ and R⁴ are respectively as defined above),and the reaction substrate represented by the formula (VI), and in orderto obtain the objective compound of high quality, these impurities needto be removed. Generally, however, any impurity structurally resemblingthe objective compound (structural analog) is not easy to remove, and inorder that such impurities may be removed to give the objective compoundof high quality, a valid purification/isolation process is required. Theinventors of the present invention found that said impurities can beefficiently removed by carrying out a crystallization under theconditions described below.

The crystallization solvent for use in the present invention ispreferably an aliphatic hydrocarbon solvent. Specifically, there can bementioned, for example, aliphatic hydrocarbons of 5 to 20 carbon atoms,such as pentane, petroleum ether, neopentane, hexane, cyclohexane,methylcyclohexane, heptane, cycloheptane, octane, isooctane, nonane,decane, undecane, dodecane, etc. Among these, pentane, hexane,methylcyclohexane, heptane, octane, and isooctane are preferred. Thesemay be used each independently or in a combination of two or morespecies.

Particularly in terms of removal of the solvent from wet crystals bydesiccation or the recovery and reuse of the solvent (distillativerecovery), the use of a solvent having a comparatively low boiling pointis preferred. As such solvents, there can generally be mentionedsolvents having a boiling point of not higher than about 100° C. atatmospheric or subatmospheric pressure. More particularly, for example,aliphatic hydrocarbon solvents of 5 to 8 carbon atoms, such as pentane,hexane, methylcyclohexane, heptane, octane, isooctane, etc. can bementioned, and when the cost of the solvent, ease of handling, and otherfactors are globally taken into consideration, hexane andmethylcyclohexane are more preferred.

The use of the above aliphatic hydrocarbon solvent provides for thestabilization and assurance of a high yield of the above compound aswell as a high degree of purification, that is to say effective removalof various impurities, particularly said compounds (XIII), (XIV), (XV)and (VI). The level of use of said aliphatic hydrocarbon solvent ispreferably such that the obtainable product at completion ofcrystallization of said compound (VII) retains sufficient fluidity, andmay, for example, be about 5 to 20 parts by weight, or even more in somecases, relative to said compound (VII).

For the crystallization of said compound (VII) in the present invention,crystallization by cooling, crystallization by concentration, and otherprocesses for crystallization can be used each independently or incombination. The crystallization by concentration, mentioned above, maybe a crystallization process in which a solution composed of a solventother than the aliphatic hydrocarbon solvent is converted to a solutioncomposed of said aliphatic hydrocarbon solvent. Moreover, seed crystalsmay be added in this crystallization.

In the present invention, for the purpose of improving at least oneparameter among the solubility, yield, treatment concentration,purification effect (efficiency of impurity removal), and physicalproperties of obtainable crystals of the above compound (VII), anauxiliary solvent can be used in addition to said aliphatic hydrocarbonsolvent in conducting the crystallization. The above auxiliary solventmay be added to said aliphatic hydrocarbon solvent as necessary or theabove compound (VII) may be dissolved in the auxiliary solvent inadvance and the solution added to said aliphatic hydrocarbon solvent.

The auxiliary solvent mentioned above is not particularly restricted butincludes, for example, acetone, methyl ethyl ketone, tetrahydrofuran,methyl tert-butyl ether, ethyl acetate, isopropanol, tert-butyl acetate,ethanol, isopropyl alcohol, toluene, benzene, xylene, chlorobenzene,methylene chloride, chloroform, and 1,2-dichloroethane, etc. These maybe used each independently or in a combination of two or more species.Among these, ethyl acetate, toluene, methyl tert-butyl ether, methylenechloride, etc. contribute to increased solubility and improved treatmenteffects such as treatment concentration and purification effect.

The auxiliary solvent mentioned above expresses its effect moreprominently when used in a suitable amount in combination with saidaliphatic hydrocarbon solvent, which suitable amount is establishedaccording to the characteristics of auxiliary solvent in relation to thedesired effect and other factors. The optimal level of use of saidauxiliary solvent can be found by simple experimentation. From thestandpoint of yield and purification effect, the level of use of theabove auxiliary solvent is preferably such that the weight ratio of saidauxiliary solvent and said aliphatic hydrocarbon solvent (auxiliarysolvent/aliphatic hydrocarbon solvent) is not greater than 1 atcompletion of the procedure for crystallization of said compound (VII).The more preferred level is such that said ratio will be 0.5 or less.

The purification/isolation method according to the present invention canbe carried out in the neighborhood of room temperature. Where necessary,the method can be practiced under warming or cooling, for example at atemperature not over about 60° C., usually at 50° C. to −30° C.

The above compound (VII) thus obtained can be separated by asolid-liquid separation technique, optionally followed by cake washingand drying. The above solid-liquid separation technique is notparticularly restricted but includes, for example, filtration underpressure, suction filtration, centrifugation, and so forth. Theabove-mentioned drying is preferably carried out under reduced pressure(drying in vacuo) at a temperature not exceeding about 60° C. in orderto avoid pyrolysis or fusion.

In the acyloxymethyldioxanylacetic acid derivative of the followingformula (VII);

as obtained in the step (4), R² and R³ each independently represents ahydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms.Specifically, methyl, ethyl, tert-butyl, hexyl, phenyl, benzyl,p-methoxybenzyl and like groups can be mentioned. The preferred is amethyl group.

Moreover, R² and R³ may jointly form a ring. For example, R² and R³ mayform a cyclopentane ring, a cyclohexane ring, a cycloheptane ring, abenzocyclopentane ring or the like to thereby constitute a spirostructure with the 1,3-dioxane ring.

Step (5)

In this step, the (4R,6S)-configured acyloxy-methyldioxanylacetic acidderivative represented by the following formula (VII);

as obtained in the step (4), is subjected to solvolysis in the presenceof a base by the known method or the like, to give a (4R,6S)-configuredhydroxymethyldioxanylacetic acid derivative of the general formula (I);

The base which can be used for the solvolysis in the step (5) includesinorganic and organic bases, such as sodium carbonate, potassiumcarbonate, sodium hydrogen carbonate, potassium hydrogen carbonate,sodium hydroxide, potassium hydroxide, calcium hydroxide, lithiumhydroxide, barium hydroxide, magnesium hydroxide, sodium acetate,potassium acetate, ammonia, triethylamine, pyridine, piperidine,N,N-dimethylaminopyridine, and so forth. The preferred is potassiumcarbonate.

The level of use of the base in this step, relative to theacyloxymethyldioxanylacetic acid derivative, is 0001 equivalent to 5equivalents, preferably 0.01 equivalent to 1.0 equivalent.

In step (5), for effecting the solvolysis, the reaction is carried outin water or in a protic organic solvent, or in a mixture of water or aprotic organic solvent and an aprotic organic solvent. The proticorganic solvent mentioned above includes, for example, alcohol solvents,such as methanol, ethanol, butanol, isopropyl alcohol, ethylene glycol,methoxyethanol, etc.; and amine solvents, such as diethylamine,pyrrolidine, piperidine, and so forth. The aprotic organic solventmentioned above includes, for example, hydrocarbon solvents such asbenzene, toluene, cyclohexane, etc.; ether solvents such as diethylether, tetrahydrofuran, 1,4-dioxane, methyl t-butyl ether,dimethoxyethane, etc.; ester solvents such as ethyl acetate, butylacetate, etc.; ketone solvents such as acetone, methyl ethyl ketone,etc.; halogenated hydrocarbon solvents such as methylene chloride,chloroform, 1,1,1-trichloroethane, etc.; nitrogen-containing solventssuch as N,N-dimethylformamide, acetonitrile, etc.; and aprotic polarsolvents such as dimethyl sulfoxide, N-methylpyrrolidone,hexamethylphosphoric triamide, and so forth. The preferred are water,methanol, and ethanol.

The reaction temperature for the step (5) is −20° C. to 100° C.,preferably −10° C. to 50° C.

The after-treatment following completion of the reaction may be theafter-treatment which is generally carried out for recovery of theproduct from the reaction mixture. For example, the reaction mixtureavailable on completion of the reaction is added with water andextracted with the common extractant solvent, such as ethyl acetate,diethyl ether, methylene chloride, toluene, hexane or the like. From theextract thus obtained, the reaction solvent and extractant solvent areremoved by heating under reduced pressure or the like procedure, toisolate the objective compound. As an alternative, after completion ofthe reaction, the reaction solvent may be immediately distilled off byheating under reduced pressure or the like procedure, and then the sameprocedure as above be carried out. The objective product thus obtainedmay be purified to a still higher purity by the routine method such aspurification by crystallization, fractional distillation, columnchromatography, and/or the like.

BEST MODE FOR CARRYING OUT THE INVENTION

The following examples are intended to illustrate the present inventionin further detail without defining the scope of the invention.

EXAMPLE 1 (5S)-5,6-dihydroxy-3-oxohexanoic tert-butyl ester

To 21.3 mL (35 mmol) of a solution of n-butyllithium in hexane (1.5mol/L) was added a solution of diisopropylamine (3.54 g, 35mmol)-tetrahydrofuran (10 mL) dropwise under stirring at 5° C., and themixture was stirred under argon for 1 hour to prepare a lithiumdiisopropylamide solution. After this solution was cooled to −70° C.,4.06 g (35 mmol) of tert-butyl acetate was added dropwise thereto andthe mixture was stirred at the same temperature for 1 hour. Then, 1 mLof a solution of (S)-β-hydroxy-γ-butyrolactone (1.02 g, 10 mmol) in THFwas added dropwise and the whole mixture was stirred at −70° C. for 2hours, at the end of which time the temperature was increased to −10° C.In a separate vessel, 60 mL of 1N-hydrochloric acid and 60 ml of diethylether were stirred to mix and the above reaction mixture was pouredtherein. The aqueous phase was adjusted to pH 6.5 with 1N-hydrochloricacid and, after standing, the organic layer was separated. The aqueouslayer was further extracted with 3 portions of ethyl acetate, 50 mLeach, and the organic layers were pooled and dehydrated over anhydrousmagnesium sulfate. The solvent was then distilled off under reducedpressure and the residue was purified by silica gel columnchromatography (Kieselgel 60, product of Merck; hexane:ethylacetate=2:1). By this procedure, 1.56 g of(5S)-5,6-dihydroxy-3-oxohexanoic tert-butyl ester (yellow oil) wasobtained. Yield: 71%.

¹H-NMR (CDCl₃, 400 MHz/ppm); 1.48 (9H, s), 2.68-2.83 (2H, m), 3.05-3.80(2H, bs), 3.42 (2H, s), 4.02-4.17 (2H, m), 4.40 (1H, m)

¹³C-NMR (CDCl₃, 400 MHz/ppm); 27.8, 45.7, 51.0, 65.6, 68.0, 82.3, 166.4,203.4

IR (neat); 3425, 3000, 1710, 850 cm⁻¹

[α]_(D) ²⁰=−17.25 (c=2.14, MeOH).

EXAMPLE 2 (5S)-5,6-dihydroxy-3-oxohexanoic tert-butyl ester

To 30 mL (45 mmol) of a solution of n-butyllithium in hexane (1.5 mol/L)was added a solution of diisopropylamine (5.01 g, 49.5mmol)-tetrahydrofuran (5 mL) dropwise under stirring at 5° C., and themixture was stirred under argon for 1 hour to prepare a lithiumdiisopropylamide solution. In a separate vessel, 1.02 g (10 mmol) of(S)-β-hydroxy-γ-butyrolactone and 2.32 g (20 mmol) of tert-butyl acetatewere dissolved in 8.0 mL of tetrahydrofuran and the mixture was stirredunder argon at 0 to 5° C. To this solution, the lithium diisopropylamidesolution prepared above was added dropwise over 30 minutes, and themixture was further stirred at 5 to 20° C. for 16 hours. In a separatevessel, 35 mL of 3N-hydrochloric acid and 30 ml of ethyl acetate werestirred to mix and the above reaction mixture was poured therein. Afterstanding, the organic layer was taken, washed with saturated aqueoussodium chloride solution, and dehydrated over anhydrous magnesiumsulfate. The solvent was then distilled off under reduced pressure andthe residue was purified by silica gel column chromatography (Kieselgel60, product of Merck; hexane:ethyl acetate=2:1) to give 124 mg of(5S)-5,6-dihydroxy-3-oxohexanoic tert-butyl ester (yellow oil). Yield6%.

EXAMPLE 3 (5S)-5,6-dihydroxy-3-oxohexanoic tert-butyl ester

To 22.9 mL (35 mmol) of a solution of n-butyllithium in hexane (1.5mol/L) was added a solution of diisopropylamine (3.90 g, 38.5mmol)-tetrahydrofuran (3 mL) dropwise under stirring at 5° C., and themixture was stirred under argon for 1 hour to prepare a lithiumdiisopropylamide solution. In a separate vessel, 1.02 g (10 mmol) of(S)-β-hydroxy-γ-butyrolactone and 2.32 g (20 mmol) of tert-butyl acetatewere dissolved in 3.0 mL of tetrahydrofuran and the mixture was stirredunder argon at 0 to 5° C. To this solution, 5.7 g (10 mmol) of asolution (1.75 mol/kg) of tert-butylmagnesium chloride intoluene/tetrahydrofuran (1:2.5 by weight) was added dropwise over 10minutes, and the mixture was further stirred at 5° C. for 50 minutes. Tothis mixture was added the above-prepared lithium diisopropylamidesolution dropwise over 30 minutes, followed by 16 hours of stirring at 5to 20° C.

In a separate vessel, 30 mL of 3N-hydrochloric acid and 30 ml of ethylacetate were stirred to mix and the above reaction mixture was pouredtherein. After standing, the organic layer was taken, washed withsaturated aqueous sodium chloride solution, and dehydrated overanhydrous magnesium sulfate. The solvent was then distilled off underreduced pressure and the residue was purified by silica gel columnchromatography (Kieselgel 60, product of Merck; hexane:ethylacetate=2:1) to give 980 mg of (5S)-5,6-dihydroxy-3-oxohexanoictert-butyl ester (red oil). Yield 48%.

EXAMPLE 4 (5S)-5,6-dihydroxy-3-oxohexanoic tert-butyl ester

To a suspension of 9.82 g (150 mmol) of zinc dust in 40 mL oftetrahydrofuran was added 1.9 mL (15 mmol) of trimethylsilyl chloride atroom temperature, and the mixture was stirred for 30 minutes. To thismixture were added 2.4 mL (10.5 mmol) of tert-butyl α-bromoacetate and4.27 g (42 mmol) of (S)-β-hydroxy-γ-butyrolactone, and the temperaturewas increased to 65° C. At the same temperature, 15.3 mL (94.5 mmol) oftert-butyl α-bromoacetate was further added gradually over 30 minutes.After completion of addition, the mixture was further stirred at 65° C.for 30 minutes, at the end of which time the reaction mixture was cooledto room temperature and diluted with 50 mL of water. The reactionmixture was then adjusted to pH 6.8 with 20% aqueous NaOH solution andthe precipitated solid was filtered off. The filtrate was extracted with3 portions of ethyl acetate, 100 mL each, and the organic layers werecombined and dehydrated over anhydrous sodium sulfate. The solvent wasthen distilled off under reduced pressure to give a yellow oil. Thisresidue was purified by silica gel column chromatography (Kieselgel 60,product of Merck; hexane: acetone=5:1) to give 2.66 g of(5S)-5,6-dihydroxy-3-oxohexanoic tert-butyl ester (yellow oil). Yield29%.

EXAMPLE 5 (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester

To a solution of 16.8 g (77 mmol) of the(5S)-5,6-dihydroxy-3-oxohexanoic tert-butyl ester produced in Example 1in 120 mL of methylene chloride were added 11.2 mL of pyridine and 10.2mL of benzoyl chloride at 0° C., and the mixture was stirred at 0° C.for 2 hours. After completion of the reaction, the reaction mixture wasdiluted with 38 mL of water and adjusted to pH 7 with 20% aqueous NaOHsolution. The aqueous layer was separated and further extracted with 2portions of methylene chloride, 120 mL each. The organic layers werecombined and dehydrated over anhydrous sodium sulfate and the solventwas distilled off under reduced pressure to give an oil. This residuewas purified by silica gel column chromatography (Kieselgel 60, productof Merck; hexane: acetone=5:1) to give 19.3 g of(5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester (whitesolid). Yield 78%.

¹H-NMR (CDCl₃, 400 MHz/ppm); 1.46 (9H, s), 2.85 (2H, d), 3.09 (1H, d),3.42 (2H, s), 4.36 (2H, m), 4.50 (1H, m), 7.45 (2H, dd), 7.56 (1H, dd),8.05 (2H, d)

IR (KBr); 3495, 1730, 1700, 1335, 1290, 1150, 720 cm⁻¹ m.p. 67 to 68° C.

EXAMPLE 6 (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester

To 109 mL (175 mmol) of a solution of n-butyllithium in hexane (1.5mol/L) was added a solution of diisopropylamine (19.48 g, 195mmol)-tetrahydrofuran (30 mL) dropwise under stirring at 5° C., and themixture was stirred under argon for 1 hour to prepare a lithiumdiisopropylamide solution. In a separate vessel, 5.10 g (50 mmol) of(S)-β-hydroxy-γ-butyrolactone and 14.5 g (125 mmol) of tert-butylacetate were dissolved in 60 ml of tetrahydrofuran and the solution wasstirred under argon at 0 to 5° C. To this solution, 27.8 g (50 mmol) ofa mixed solution of tert-butylmagnesium chloride intoluene/tetrahydrofuran (1:2.5, by weight) (1.8 mol/kg) was addeddropwise over 30 minutes and the mixture was further stirred at 5° C.for 30 minutes. To this mixture was added the above-prepared lithiumdiisopropylamide solution dropwise over 3 hours, and the mixture wasfurther stirred at 5 to 20° C. for 16 hours. In a separate vessel, 25.05g of acetic acid, 75 mL of water, and 150 mL of ethyl acetate werestirred to mix and the above reaction mixture was poured therein. Afterstanding, the aqueous layer was separated and further extracted with 2portions of ethyl acetate, 150 mL each. The organic layers werecombined, diluted with 20 mL of saturated aqueous sodium chloridesolution, and adjusted to pH 3 with 3N-hydrochloric acid. After theaqueous layer was separated, the organic layer was further washed with20 mL of saturated aqueous sodium hydrogen carbonate solution anddehydrated over anhydrous magnesium sulfate. The solvent was distilledoff under reduced pressure to give 18.18 g of a yellow oil containing(5S)-5,6-dihydroxy-3-oxohexanoic tert-butyl ester.

To the above oil, 6.32 g (80 mmol) of pyridine and 50 mL of toluene wereadded, and the mixture was cooled to 5° C. To this mixture was added6.32 g (45 mmol) of benzoyl chloride, and the whole mixture was stirredat 5° C. for 1.5 hours. Then, 25 mL of water and 15 mL of3N-hydrochloric acid were added. This mixture was extracted with 100 mLof ethyl acetate and the organic layer was washed with 30 mL ofsaturated sodium hydrogen carbonate solution and 50 mL of water twice.The solvent was then distilled off under reduced pressure to give 18.43g of a yellow oil. This oil was analyzed by high-performance liquidchromatography (column: Develosil ODS-HG-3 4.6×250 mm, product of NomuraChemical, eluent: water/acetonitrile=50/50, flow rate: 1.0 mL/min,detector: UV 220 nm, column temperature: 40° C.). The analysis showedthat the reaction yield of (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoictert-butyl ester was 55%.

EXAMPLE 7 (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester

To 125 mL (225 mmol) of a solution of n-butylmagnesium chloride intetrahydrofuran (1.8 mol/L) was added 25.04 g (247.5 mmol) ofdiisopropylamine dropwise under stirring at 40° C., and the mixture wasfurther stirred under argon at 40° C. for 2 hours to prepare a whiteslurry of chloromagnesium diisopropylamide. In a separate vessel, 5.10 g(50 mmol) of (S)-β-hydroxy-γ-butyrolactone and 1.45 g (125 mmol) oftert-butyl acetate were dissolved in 30 mL of dimethoxyethane and thesolution was stirred at 0 to 5° C. under argon. To this solution wasadded the above-prepared chloromagnesium diisopropylamide slurrydropwise over 3 hours, and the mixture was further stirred at 5 to 20°C. for 16 hours.

In a separate vessel, 28.4 g of acetic acid, 100 mL of water, and 150 mlof ethyl acetate were stirred to mix and the above reaction mixture waspoured therein. After standing, the aqueous layer was separated andfurther extracted with 2 portions of ethyl acetate, 150 ml each. Theorganic layers were combined, diluted with 20 mL of saturated aqueoussodium chloride solution, adjusted to pH 3 with 3N-hydrochloric acid,and the aqueous layer was separated. The organic layer was furtherwashed with 20 mL of saturated aqueous sodium hydrogen carbonatesolution and dehydrated over anhydrous magnesium sulfate, and thesolvent was distilled off under reduced pressure to give 14.24 g of ared oil containing (5S)-5,6-dihydroxy-3-oxohexanoic tert-butyl ester. Tothe above oil, 6.32 g (80 mmol) of pyridine and 50 mL of toluene wereadded, and the mixture was cooled to 5° C. To this mixture was added5.62 g (40 mmol) of benzoyl chloride, and the whole mixture was stirredat 5° C. for 1 hour. Then, 25 mL of water and 15 mL of 3N-hydrochloricacid were added. This mixture was extracted with 150 mL of ethyl acetateand the organic layer was washed with 30 mL of saturated sodium hydrogencarbonate solution and 30 mL of water twice. The solvent was thendistilled off under reduced pressure to give 18.12 g of a yellow oil.This oil was analyzed by high-performance liquid chromatography (column:Develosil ODS-HG-3 4.6×250 mm, product of Nomura Chemical, eluent:water/acetonitrile=50/50, flow rate: 1.0 mL/min, detector: UV 220 nm,column temperature: 40° C.). As a result, the reaction yield of(5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester was found tobe 53%.

EXAMPLE 8 (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester

To 141 mL (225 mmol) of a solution of n-butyllithium in hexane (1.6mol/L) was added a solution of diisopropylamine (25.04 g, 247.5mmol)-tetrahydrofuran (30 mL) dropwise under stirring at 5° C., and themixture was stirred under argon for 1 hour to prepare a lithiumdiisopropylamide solution. In a separate vessel, 5.10 g (50 mmol) of(S)-β-hydroxy-γ-butyrolactone, 14.5 g (125 mmol) of tert-butyl acetateand 9.52 g (100 mmol) of anhydrous magnesium chloride were dissolved in30 ml of tetrahydrofuran and the solution was stirred under argon at 0to 5° C. To this solution was added the above-prepared a lithiumdiisopropylamide solution dropwise over 3 hours, and the mixture wasfurther stirred at 5 to 20° C. for 16 hours. In a separate vessel, 28.4g of acetic acid, 100 mL of water, and 150 mL of ethyl acetate werestirred to mix and the above reaction mixture was poured therein. Afterstanding, the aqueous layer was separated and further extracted with 2portions of ethyl acetate, 150 ml each. The organic layers werecombined, diluted with 20 mL of saturated aqueous sodium chloridesolution, adjusted to pH 3 with 3N-hydrochloric acid, and the aqueouslayer was separated. The organic layer was further washed with 20 mL ofsaturated aqueous sodium hydrogen carbonate solution and dehydrated overanhydrous magnesium sulfate, and the solvent was distilled off underreduced pressure to give 12.74 g of a red oil containing(5S)-5,6-dihydroxy-3-oxohexanoic tert-butyl ester.

To the above oil, 6.32 g (80 mmol) of pyridine and 50 mL of toluene wereadded, and the mixture was cooled to 5° C. To this mixture was added5.62 g (40 mmol) of benzoyl chloride, and the whole mixture was stirredat 5° C. for 1 hour. Then, 25 mL of water and 15 mL of 3N-hydrochloricacid were added. This mixture was extracted with 150 mL of ethyl acetateand the organic layer was washed with 30 mL of saturated sodium hydrogencarbonate solution and 30 mL of water twice. The solvent was thendistilled off under reduced pressure to give 17.92 g of a red oil. Thisoil was analyzed by high-performance liquid chromatography (column:Develosil ODS-HG-3 4.6×250 mm, product of Nomura Chemical, eluent:water/acetonitrile=50/50, flow rate: 1.0 mL/min, detector: UV 220 nm,column temperature: 40° C.). As a result, the reaction yield of(5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester was found tobe 55%.

EXAMPLE 9 Purification of (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoictert-butyl ester

The oil containing (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butylester as produced in Example 6 was analyzed high-performance liquidchromatography (the conditions are described in Example 6). Purity: 48.2weight % (58.1 area %). As an impurity, the oil contained 4.8 weight %(6.5 area %) of (5S)-5,6-dibenzyloxy-3-oxohexanoic tert-butyl ester. To18.43 g of this oil ((5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoictert-butyl ester: 8.88 g) was added 30 mL of toluene to make ahomogeneous solution, followed by addition of 80 mL of hexane, and themixture was cooled to 5° C. (treatment concentration: 8% (substrateweight/solution volume)). To this opaque solution, about 10 mg of seedcrystals were added, and the mixture was further stirred vigorously atthe same temperature for 1 hour. The resulting crystals were collectedby suction filtration, drained thoroughly, washed with 50 mL of hexane,and dried in vacuo (ca 1-5 mmHg, 20 to 40° C., 2 hours), whereby 6.12 gof (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester wasobtained as crystals (crystallization recovery rate 67%). Analysis:purity 97.7 weight % (95.4 area %); (5S)-5,6-dibenzyloxy-3-oxohexanoictert-butyl ester content: 0.7 weight % (0.7 area %)

EXAMPLE 10 Purification of (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoictert-butyl ester

The oil containing (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butylester as produced in Example 7 was analyzed by high-performance columnchromatography (the conditions are described in Example 6). The puritywas 45.0 weight % (47.2 area %) and the impurity(5S)-5,6-dibenzyloxy-3-oxohexanoic tert-butyl ester content: 4.7 weight% (4.9 area %). To 18.12 g of this oil((5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester: 8.15 g) wasadded 20 mL of toluene to prepare a homogeneous solution. To thissolution was added 80 mL of hexane, and the mixture was cooled to −30°C. (treatment concentration: 8% (substrate weight/solution volume)). Tothe resulting opaque solution was added about 10 mg of seed crystals,and the mixture was stirred vigorously at the same temperature for 1hour. The crystals separating out were collected by suction filtration,drained thoroughly, and washed with 50 mL of hexane. This crystal cropwas dried in vacuo (ca 1 to 5 mmHg, 20 to 40° C., 2 hours) to obtain6.68 g of (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl estercrystals (crystallization recovery rate 77%). Analysis of the crystalsshowed a purity of 95.8 weight % (94.9 area %) and a(5S)-5,6-dibenzyloxy-3-oxohexanoic tert-butyl ester content of 1.6weight % (1.6 area %).

EXAMPLE 11 (3R,5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester

A large test tube was charged with 5 ml of Medium A describedhereinbefore and, after sterilization, inoculated with one of themicroorganisms indicated in Table 1 and Table 2. Aerobic shake culturewas carried out at 27° C. for 2 to 3 days. From a 1.5 ml portion of theresulting culture, the cells were harvested by centrifugation andsuspended in 0.5 ml of 100 mM phosphate buffer (pH 6.5) containing 0.05%of (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester and 8% ofglucose. The suspension was put in a test tube equipped with a threadedstopper and the reaction was carried out under shaking at 27° C. for 20hours. After the reaction, 4 volumes of ethyl acetate were added to thereaction mixture and after thorough mixing, the cells were centrifugallyremoved. The supernatant was analyzed by high-performance liquidchromatography for the amount of(3R,5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced perml of the reaction mixture and the diastereomer ratio (=(3R,5S)/(3S,5S)ratio). The results are shown in Table 1 and Table 2. TABLE 1 OutputD.E. Microorganism (ug/ml) (%) Ashbya gossypii IFO 0560 500.0 61.1Botryoascus synnaedendrus IFO 1604 330.6 63.7 Brettanomyces custersianusIFO 1585 59.7 95.1 Candida arborea IAM 4147 77.3 72.4 Candida catenulataIFO 0745 46.5 10.1 Candida fennica CBS 6028 135.1 87.8 Candida galactaIFO 10031 121.0 84.3 Candida haemulonii IFO 10001 133.7 94.3 Candidamagnoliae IFO 0705 175.0 27.1 Candida musae IFO 1582 39.8 33.5 Candidanitratophila IFO 10004 20.1 48.3 Candida parapsilosis IFO 0585 118.465.7 Candida pararugosa IFO 0966 26.6 56.0 Candida stellata IFO 070127.4 100.0 Citeromyces matritensis IFO 0651 446.9 26.2 Clavisporalusitaniae IFO 1019 342.1 53.7 Cryptococcus laurentii IFO 0609 37.5 14.5Debaryomyces carsonii IFO 0795 449.1 97.3 Debaryomyces hansenii var.fabryi IFO 0794 55.4 78.8 Debaryomyces hansenii var. hansenii IFO 0032130.1 93.6 Debaryomyces hansenii var. hansenii IFO 0047 135.4 95.4Debaryomyces hansenii var. hansenii IFO 0018 103.1 95.5 Debaryomyceskloeckeri 140.7 95.4 Debaryomyces marama IFO 0668 161.5 95.4Debaryomyces pseudopolymorphus IFO 1026 75.4 90.9 Debaryomycesrobertsiae IFO 1277 278.7 66.1 Debaryomyces sp. IFO 0025 32.7 74.7Dekkera anomala IFO 0627 115.8 94.9 Dipodascus armillariae IFO 0102154.6 76.8 Dipodascus ovetensis IFO 1201 134.9 96.9 Dipodascustetrasperma CBS 765.70 203.0 33.1 Galactomyces reessii CBS 179.60 378.825.0 Geotrichum candidum CBS 187.67 148.6 51.8 Geotrichum fermentans IFO1199 98.4 93.9 Geotrichum fragrans CBS 164.32 29.1 94.1 Geotrichumloubieri CBS 252.61 81.4 14.8 Hanseniaspora guilliermondii IAM 4972 35.84.0 Hansenula methanolosa 93.7 99.2 Hansenula polymarpha DL1 AKU 475221.6 100.0 Hormoascus philentomus IFO 1847 176.6 84.2 Hormoascusplatypodis IFO 1471 260.8 36.6 Hyphopichia burtonii IFO 0844 228.8 77.7Issatchenkia orientalis IFO 1279 443.0 36.4 Issatchenkia terricola IFO0933 258.4 100.0 Kluyveromyces lactis IFO 1012 324.6 48.8 Kluyveromycesmarxianus IFO 0541 102.7 85.9 Kluyveromyces marxianus IFO 0288 419.327.1 Kluyveromyces polysporus IFO 0996 132.6 5.1 Kluyveromycesthermotolerans IFO 0662 500.0 100.0 Komagataella pastoris IFO 1013 246.066.4 Linomyces starkeyi IFO 0678 28.6 100.0 Metschnikowia bicuspidataIFO 1408 381.0 39.6 Metschnikowia pulcherrima IFO 0561 359.4 480

TABLE 2 Output D.E. Microorganism (ug/ml) (%) Nakazawaea holstii IFO0980 0.5 100.0 Ogataeaminuta var. minuta IFO 0975 18.2 9.4 OgataeapiniIFO 1342 268.3 43.1 Ogataea polymorpha IFO 0799 500.0 67.3 Ogataeapolymorpha IFO 1475 275.0 6.0 Ogataea wickerhamii IFO 1706 144.8 73.6Pachysolen tannophilus IFO 1007 488.2 5.4 Pichia canadensis IFO 097617.5 86.9 Pichia farinosa IAM 4369 208.9 71.4 Pichia jandinii IFO 0987296.4 96.9 Pichia Saitoi IAM 4945 97.4 22.5 Pichia toletana IFO 0950300.0 13.1 Pichia triangularis IFO 0836 328.8 24.6 Pichia wickerhamiiIFO 1278 175.8 81.7 Rhodotorula graminis IFO 0190 96.5 3.3 Rhodotorulaminuta IFO 0387 108.3 12.0 Rhodotorula minuta IFO 0715 0.5 100.0Rhodsporidium diobovatum IFO 0688 1.8 17.4 Rhodsporidium toruloides IFO0413 10.2 46.7 Saccharomyces bayanus IFO 0251 375.2 18.3 Saccharomycespastorianus IFO 1265 442.5 80.5 Saccharomyces pastorianus ATCC 9080 83.172.9 Saccharomyces rosei IFO 0252 456.8 83.1 Saccharomyces sake 349.592.6 Saccharomyces steineri IAM 4608 98.3 100.0 Saccharomyces unisporusIFO 0215 97.0 84.5 Saccharomycodes ludwigii IFO 0339 99.0 43.8Saccharomycopsis capsularis IFO 0672 112.5 76.8 Saccharomycopsis malangaIFO 1710 0.3 100.0 Saturnospora dispora IFO 0035 16.6 16.8Schizoblastosporion kobayasii IFO 1644 207.0 54.6 Schizosaccharomycespombe IFO 0347 119.5 55.2 Schizosaccharomyces pombe IFO 0362 96.3 56.0Schwanniomyces occidentalis var. IFO 1840 219.7 46.9 occidentalisSporidiobolus johnsonii IFO 6903 2.7 100.0 Sporobolomyces pararoseus IFO0471 66.0 67.8 Sporobolomyces salmonicolor IFO 1038 8.8 100.0Torulaspora delbrueckii IFO 0381 186.4 95.9 Torulopsis methanolevescens337.0 33.2 Torulopsis osboenis IFO 0646 58.5 16.9 Torulopsis sp. 99.784.9 Torulopsis uvae IFO 0649 287.1 88.8 Trichosporon pullulans 20.951.0 Trichosporon sp. 4.6 19.2 Trigonopsis variabilis IFO 0671 126.413.4 Willopsis saturnus var. mrakii IFO 0895 445.3 3.6 Willopsissaturnus var. saturnus IFO 0992 394.4 6.1 Yamadazyma farinosa IFO 0459472.7 86.7 Yamadazyma farinosa IFO 0602 97.0 55.0 Yamadazyma haplophilaIFO 0947 7.2 66.4 Zygosaccharomyces naniwensis IFO 0524 263.1 43.0Zygosaccharomyces sp. IFO 0522 282.8 9.4

EXAMPLE 12 (3R,5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester

Using 5 ml of Medium B described hereinbefore, the microorganismsindicated in Table 3 were cultured in the same manner as in Example 11.Thereafter, the reaction was carried out in the same manner. The resultsare shown in Table 3. TABLE 3 Output D.E. Microorganism (ug/ml) (%)Acidiphilium cryptum IFO 14242 2.7 12.2 Aerobacter cloacae IAM 1221 29.279.0 Alcaligenes xylosoxidans IFO 13495 13.2 38.9 Alcaligenesxylosoxidans subsp. IFO 12669 3.9 22.9 denitrificans Alcaligenesxylosoxidans subsp. ATCC 15173 11.2 90.3 denitrificans Arthrobacterglobiformis ATCC 8010 0.4 100.0 Arthrobacter protophormiae IFO 1212828.1 100.0 Aureobacterium esteraromaticum IFO 3752 245.9 24.1 Bacillusbadius IAM 11059 0.3 100.0 Bacillus sphaericus IFO 3525 0.7 20.2Brevibacterium ammomiagenes IFO 12071 193.1 98.8 Buttiauxella agrestisJCM 1090 25.7 40.3 Cedecea davisiae JCM 1685 3.2 37.0 Cellulomonas sp.JCM 2471 119.8 95.9 Cellulomonas turbata IFO 15015 88.8 24.3 Citrobacterfreundii IFO 12681 75.2 68.9 Clostridium cylindrosporum IFO 13695 4.850.6 Comamonas testosteroni IFO 12047 1.1 48.8 Corynebacteriumacectoacidophilum ATCC 21476 211.3 63.4 Corynebacterium ammoniagenes IFO12072 1.8 66.0 Corynebacterium glutamicum ATCC 21269 269.3 92.2Corynebacterium glutamicus ATCC 13287 276.9 98.3 Enterobacter aerogenesIFO 13534 54.5 91.4 Enterobacter cloacae IFO 12935 490.7 88.9 Erwiniacarotovora subsp. carotovora IFO 3830 1.1 100.0 Escherichia coli IFO12734 23.2 53.9 Flavobacterium flavcsccus 17.3 25.1 Klebsiellaplanticola IFO 3317 127.3 61.6 Luteococcus japonicus IFO 12422 0.2 100.0Microbacterium arborescens IFO 3750 7.6 90.8 Micrococcus flavus 4.0 30.7Micrococcus luteus IFO 13867 500.0 13.5 Ochrobactrum sp. IFO 12950 12.551.4 Proteus inconstans IFO 12931 5.6 87.6 Proteus mirabilis IFO 38491.1 100.0 Proteus rettgeri IFO 13501 0.3 100.0 Proteus vulgaris IFO 31670.5 100.0 Providencia stuartii IFO 12930 2.9 76.9 Pseudomonas acruginosaIAM 1007 2.8 100.0 Pseudomonas putida IFO 14164 11.2 31.1 Pseudomonasstutzeri IFO 13596 8.0 32.5 Rhodococcus equi JCM 1313 48.4 72.7 Sarcinalutea 369.2 86.8 Serratia plymuthicum IFO 3055 2.7 100.0 Serratiaproteamaculans subsp. IFO 12979 38.5 47.3 proteamaculansSphingobacterium spiritivorum JCM 1277 61.9 33.6 Tsukamurellapaurometabolum IFO 12160 40.6 8.2

EXAMPLE 13 (3R,5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester

Using 5 ml of Medium C described hereinbefore, the microorganismsindicated in Table 4 were cultured in the same manner as in Example 11.From 5 ml of each culture, the cells were centrifugally harvested,suspended in 0.5 ml of 100 mM phosphate buffer (pH 6.5) containing 0.05%of (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester and 8% ofglucose, and the reaction was carried out in the same manner. Theresults are shown in Table 4. TABLE 4 Output D.E. Microorganism (ug/ml)(%) Absidia orchidis HUT 1036 4.4 25.2 Acremonium bacillisporum IFO 93871.0 100.0 Aegerita candida IFO 6988 500.0 92.8 Agrocybe cylindracea IFO30299 96.2 59.5 Amylostereum areolatum IFO 9221 71.7 32.9 Aspergillusparasiticus IFO 4403 2.6 100.0 Aspergillus phoenicis IFO 6670 1.4 32.0Byssochlamys fulva IFO 6307 164.6 99.2 Chaetomidium fimeti IFO 30419 0.5100.0 Chaetosartorya stromatoides IFO 9652 1.6 100.0 Cladosporiumresinae F. avellaneum IFO 6367 1.4 100.0 Coprinus cinereus 401.3 76.0Coprinus lagopus IFO 9533 37.9 93.7 Coprinus sp. 1.9 100.0 Crinipellisstipitaria IFO 30259 16.6 40.6 Endophragmia alternata IFO 30204 10.454.9 Flavolus arcularius 217.0 8.8 Fomitopsis pubertatis 102.0 6.6Fusarium merismoides IFO 30040 125.7 16.2 Ganoderma lucidum IFO 318632.1 31.8 Glomerella cingulata IFO 5257 47.8 78.7 Laetiporus sulphureusII66.9 32.8 Lentinus lepideus TD-832 165.2 35.1 Lenzites betulina IFO 8715155.9 34.5 Macrophoma commelinae IFO 9569 210.2 99.3 Monascus purpureusIFO 5965 135.7 13.7 Mortierella isabellina IFO 7829 8.7 100.0Paecilomyces varioti HUT 4028 34.6 100.0 Penicillium chermesinum IFO5800 39.9 86.7 Penicillium chrysogenum IFO 4640 133.8 97.4 Penicilliumexpansum IFO 5854 4.5 51.1 Penicillium lilacinium IFO31914 47.2 95.1Phialophora fastigiata IFO 6850 38.4 89.4 Pholiota aurivella IFO 3026574.5 100.0 Pholiota limonella IFO 31868 0.8 100.0 Pleurotus dryinus123.9 26.0 Pleurotus ostreatus 159.1 22.4 Pleurotus porrigens 247.7 87.3Scopulariopsis brevicaulis IFO 4843 88.6 23.9 Sehizophyllum commune IFO6503 119.9 43.7 Sporotrichum aurantiacum IFO 9381 84.4 8.9 Zygorhynchusmoelleri HUT 1305 167.6 93.5

EXAMPLE 14 (3R,5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester

Using 5 ml of Medium D described hereinbefore, the microorganismsindicated in Table 5 were cultured in the same manner as in Example 13.Then, the same reaction was carried out. The results are shown in Table5. TABLE 5 Output D.E. Microorganism (ug/ml) (%) Microtetrasporaroseoviolacea IFO 14098 27.8 15.4 Streptomyces achromogenes subsp. IFO14000 1.6 19.3 rubradiris Streptomyces sp. 29.4 42 Streptomyces aureusNIHJ 122 1 100

EXAMPLE 15 (3R,5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester

A Sakaguchi flask of 500 ml capacity was charged with 100 ml of a mediumcomposed of Bacto-tryptone 1.6%, Bactoyeast extract 1%, and sodiumchloride 1% (pH 7.0) and, after sterilization, inoculated withEscherichia coli HB101 (pNTCRG); FERM BP-6898 (deposited with NationalInstitute of Bioscience and Human-Technology (1-3, Higashi 1-chome,Tsukuba-shi, Ibaraki, Japan) as of Sep. 28, 1999). Shake culture wascarried out at 37° C. for 12 hours. After completion of cultivation, 1 gof (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester, 610 mg ofglucose, and 3 mg of oxidized nicotinamide-adenine dinucleotidephosphate were added and the reaction was conducted for 24 hours, duringwhich the pH was maintained at 6.5 with sodium hydroxide. Aftercompletion of the reaction, the cells were centrifugally removed and thesupernatant was extracted with 2 portions of ethyl acetate, 100 mL each.The organic phase obtained was dehydrated over anhydrous sodium sulfateand the solvent was distilled off under reduced pressure to give 900 mgof 6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester as oil. Asanalyzed by the method described in Example 11, the diastereomer ratioof this product was (3R,5S)/(3S,5S)=99.5/0.5.

¹H-NMR (CDCl₃, 400 MHz/ppm); 1.47 (9H, s), 1.63-1.82 (2H, m), 2.45 (2H,d), 4.1-4.3 (4H, m), 7.32-7.7 (3H, m), 8.0-8.22 (2H, m)

IR (neat); 3450, 3000, 1730, 1040, 850, 720 cm⁻¹

EXAMPLE 162-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester

To a solution composed of 8.94 g (27.6 mmol) of(5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced inExample 15, 35.8 mL of 2,2-dimethoxypropane, and 2.5 mL of methylenechloride was added 269 mg (1.4 mmol) of p-toluenesulfonic acid.1H₂O, andthe mixture was stirred at 20° C. for 4 hours, after which 500 mL ofsaturated sodium hydrogen carbonate solution was added. The aqueouslayer was separated and further extracted with 2 portions of methylenechloride, 20 mL each, and the organic layers were combined. The combinedsolution was dehydrated over anhydrous sodium sulfate and the solventwas distilled off under reduced pressure to give a colorless oil. Thisresidue was purified by silica gel column chromatography (Kieselgel 60,product of Merck; hexane: acetone=10:1) to give 7.24 g of2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl] acetictert-butyl ester (white solid). Yield: 72%. ¹H-NMR (CDCl₃, 400 MHz/ppm);1.44 (9H, s), 1.45 (6H, d), 1.55-1.59 (2H, m), 2.35-2.46 (2H, m),4.22-4.37 (4H, m), 7.43-7.59 (3H, m), 8.0-8.1 (2H, m)

IR (neat); 2975, 1720, 1270, 1150, 1100, 718 cm⁻¹ m.p. 55 to 56° C.

EXAMPLE 172-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester

To a solution composed of 108 mg (90.2 weight %, 0.3 mmol) of the(5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced inExample 15, 62.4 mg (0.6 mmol) of 2,2-dimethoxypropane, and 5 mL ofacetone was added 5.7 mg (0.03 mmol) of p-toluenesulfonic acid.1H₂O, andthe mixture was stirred at 40° C. for 16 hours. This reaction mixturewas analyzed by high-performance liquid chromatography (column:Develosil ODS-HG-3 4.6×250 mm, product of Nomura Chemical, eluent:water/acetonitrile=50/50, flow rate: 1.0 mL/min, detector: UV 220 nm,column temperature: 40° C.). The compositional yield values were asfollows.

2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester: 78.8%; (5S)-6-benzoyloxy-3,5-dihydroxyhexanoictert-butyl ester: 7.5%;(2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: 5.9%;2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methylester: 3.0%.

EXAMPLE 182-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester

To a solution composed of 108 mg (90.2 weight %, 0.3 mmol) of the(5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced inExample 15, 62.4 mg (0.6 mmol) of 2,2-dimethoxypropane, and 5 mL ofacetone were added 5.7 mg (0.03 mmol) of p-toluenesulfonic acid.1H₂O and11.9 mg (0.15 mmol) of pyridine, and the mixture was stirred at 40° C.for 16 hours. This reaction mixture was analyzed by high-performanceliquid chromatography (column: Develosil ODS-HG-3 4.6×250 mm, product ofNomura Chemical, eluent: water/acetonitrile=50/50, flow rate: 1.0mL/min, detector: UV 220 nm, column temperature: 40° C.). Thecompositional yield values were as follows.

2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester: 93.1%; (5S)-6-benzoyloxy-3,5-dihydroxyhexanoictert-butyl ester: 3.4%;(2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: 0.1%;2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methylester: 0.1%.

EXAMPLE 192-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester

To a solution composed of 108 mg (90.2 weight %, 0.3 mmol) of the(5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced inExample 15, 62.4 mg (0.6 mmol) of 2,2-dimethoxypropane, and 5 mL ofacetone were added 5.7 mg (0.03 mmol) of p-toluenesulfonic acid.1H₂O and15.2 mg (0.15 mmol) of triethylamine, and the mixture was stirred at 40°C. for 16 hours. This reaction mixture was analyzed by high-performanceliquid chromatography (column: Develosil ODS-HG-3 4.6×250 mm, product ofNomura Chemical, eluent: water/acetonitrile=50/50, flow rate: 1.0mL/min, detector: UV 220 nm, column temperature: 40° C.). Thecompositional yield values were as follows.

2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester: 93.3%; (5S)-6-benzoyloxy-3,5-dihydroxyhexanoictert-butyl ester: 3.0%;(2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: 0.1%;2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methylester: 0.1%.

EXAMPLE 202-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester

To a solution composed of 108 mg (90.2 weight %, 0.3 mmol) of the(5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced inExample 15, 62.4 mg (0.6 mmol) of 2,2-dimethoxypropane, and 5 mL ofacetone were added 5.7 mg (0.03 mmol) of p-toluenesulfonic acid.1H₂O and10.2 mg (0.15 mmol) of imidazole, and the mixture was stirred at 40° C.for 16 hours. This reaction mixture was analyzed by high-performanceliquid chromatography (column: Develosil ODS-HG-3 4.6×250 mm, product ofNomura Chemical, eluent: water/acetonitrile=50/50, flow rate: 1.0mL/min, detector: UV 220 nm, column temperature: 40° C.). Thecompositional yield values were as follows.

2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester: 93.9%; (5S)-6-benzoyloxy-3,5-dihydroxyhexanoictert-butyl ester: 3.0%;(2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: 0.1%;2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methylester: 0.1%.

EXAMPLE 212-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester

To a solution composed of 108 mg (90.2 weight %, 0.3 mmol) of the(5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced inExample 15, 62.4 mg (0.6 mmol) of 2,2-dimethoxypropane, and 5 mL ofacetone were added 5.7 mg (0.03 mmol) of p-toluenesulfonic acid.1H₂O and14.0 mg (0.15 mmol) of 3-methylpyridine, and the mixture was stirred at40° C. for 16 hours. This reaction mixture was analyzed byhigh-performance liquid chromatography (column: Develosil ODS-HG-34.6×250 mm, product of Nomura Chemical, eluent:water/acetonitrile=50/50, flow rate: 1.0 mL/min, detector: UV 220 nm,column temperature: 40° C.). The compositional yield values were asfollows.

2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester: 93.8%; (5S)-6-benzoyloxy-3,5-dihydroxyhexanoictert-butyl ester: 2.8%;(2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: 0.1%;2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methylester: 0.1%.

EXAMPLE 222-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester

To a solution composed of 108 mg (90.2 weight %, 0.3 mmol) of the(5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced inExample 15, 62.4 mg (0.6 mmol) of 2,2-dimethoxypropane, and 5 mL ofacetone were added 5.7 mg (0.03 mmol) of p-toluenesulfonic acid.1H₂O and18.3 mg (0.15 mmol) of N,N-dimethylaminopyridine, and the mixture wasstirred at 40° C. for 16 hours. This reaction mixture was analyzed byhigh-performance liquid chromatography (column: Develosil ODS-HG-34.6×250 mm, product of Nomura Chemical, eluent:water/acetonitrile=50/50, flow rate: 1.0 mL/min, detector: UV 220 nm,column temperature: 40° C.). The compositional yield values were asfollows.

2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester: 93.8%; (5S)-6-benzoyloxy-3,5-dihydroxyhexanoictert-butyl ester: 2.3%;(2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: 0.1%;2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methylester: 0.1%.

EXAMPLE 232-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester

To a solution composed of 108 mg (90.2 weight %, 0.3 mmol) of the(5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced inExample 15, 62.4 mg (0.6 mmol) of 2,2-dimethoxypropane, and 5 mL ofacetone were added 3.0 mg (0.03 mmol) of methanesulfonic acid and 11.9mg (0.15 mmol) of pyridine, and the mixture was stirred at 40° C. for 16hours. This reaction mixture was analyzed by high-performance liquidchromatography (column: Develosil ODS-HG-3 4.6×250 mm, product of NomuraChemical, eluent: water/acetonitrile=50/50, flow rate: 1.0 mL/min,detector: UV 220 nm, column temperature: 40° C.). The compositionalyield values were as follows.

2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester: 95.6%; (5S)-6-benzoyloxy-3,5-dihydroxyhexanoictert-butyl ester: 2.6%;(2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: 0.1%;2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methylester: undetected.

EXAMPLE 242-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester

To a solution composed of 108 mg (90.2 weight %, 0.3 mmol) of the(5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced inExample 15, 62.4 mg (0.6 mmol) of 2,2-dimethoxypropane, and 5 mL ofacetone were added 3.5 mg (0.03 mmol) of trifluoroacetic acid and 11.9mg (0.15 mmol) of pyridine, and the mixture was stirred at 40° C. for 16hours. This reaction mixture was analyzed by high-performance liquidchromatography (column: Develosil ODS-HG-3 4.6×250 mm, product of NomuraChemical, eluent: water/acetonitrile=50/50, flow rate: 1.0 mL/min,detector: UV 220 nm, column temperature: 40° C.). The compositionalyield values were as follows.

2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester: 89.3%; (5S)-6-benzoyloxy-3,5-dihydroxyhexanoictert-butyl ester: 8.8%;(2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: 0.1%;2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methylester: undetected.

EXAMPLE 252-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester

To a solution composed of 108 mg (90.2 weight %, 0.3 mmol) of the(5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced inExample 15, 62.4 mg (0.6 mmol) of 2,2-dimethoxypropane, and 5 mL ofacetone were added 1.5 mg (0.015 mmol) of sulfuric acid and 11.9 mg(0.15 mmol) of pyridine, and the mixture was stirred at 40° C. for 16hours. This reaction mixture was analyzed by high-performance liquidchromatography (column: Develosil ODS-HG-3 4.6×250 mm, product of NomuraChemical, eluent: water/acetonitrile=50/50, flow rate: 1.0 mL/min,detector: UV 220 nm, column temperature: 40° C.). The compositionalyield values were as follows.

2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester: 95.8%; (5S)-6-benzoyloxy-3,5-dihydroxyhexanoictert-butyl ester: 2.3%;(2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: 0.1%;2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methylester: undetected.

EXAMPLE 26 Purification of2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester

To a solution composed of 3.24 g (90.2 weight %, 9.0 mmol) of the(5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced inExample 15, 2.12 g (18.0 mmol) of 2,2-dimethoxypropane, and 5 mL ofacetone was added 95 mg (0.45 mmol) of p-toluenesulfonic acid.1H₂O, andthe mixture was stirred at 40° C. for 4 hours. The solvent was distilledoff under reduced pressure and the residue was extracted using 25 mL ofethyl acetate and 10 mL of saturated aqueous sodium hydrogen carbonate.After separation of the aqueous layer, the organic layer was furtherwashed with 10 mL of water. The solvent was then distilled off underreduced pressure to give 3.764 g of a colorless oil. This oil wasanalyzed by high-performance liquid chromatography (column: DevelosilODS-HG-3 4.6×250 mm, product of Nomura Chemical, eluent:water/acetonitrile=50/50, flow rate: 1.0 mL/min, detector: UV 220 nm,column temperature: 40° C.). The compositional yield values were asfollows.

2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester: 79.7 weight %. As impurities,(5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester: 0.1 weight %;(2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: 0.1weight %;2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methylester: 5.0 weight %;2-[(4S,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl)acetictert-butyl ester:0.2 weight % were contained.

To the above oil was added 30 mL of hexane, and the mixture was cooledto −30° C. (treatment concentration: 10% (substrate weight/solutionvolume)). About 10 mg of seed crystals were added and the mixture wasfurther stirred vigorously at the same temperature for 1 hour. Thecrystals separating out were collected by suction filtration, drainedthoroughly, and washed with 10 mL of cold hexane. The crystal crop wasthen dried in vacuo (ca 1 to 5 mmHg, 20 to 40° C., 2 hours) to obtain2.46 g of2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester as crystals (crystallization recovery rate 81%).Analysis of the above crystals showed a purity of 97.2 weight % (96.8area %). As impurities, the crystal crop contained:2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methylester: 2.8 weight % (2.8 area %);(5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester: undetected;(2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran:undetected;2-[(4S,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester: undetected.

EXAMPLE 27 Purification of2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester

To a solution composed of 3.24 g (90.2 weight %, 9.0 mmol) of the(5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced inExample 15, 2.12 g (18.0 mmol) of 2,2-dimethoxypropane, and 5 mL ofacetone was added 95 mg (0.45 mmol) of p-toluenesulfonic acid.1H₂O, andthe mixture was stirred at 40° C. for 4 hours. The solvent was thendistilled off under reduced pressure and the residue was extracted using25 mL of ethyl acetate and 10 mL of saturated aqueous sodium hydrogencarbonate. After separation of the aqueous layer, the organic layer wasfurther washed with 10 mL of water. The solvent was then distilled offunder reduced pressure to give 3.764 g of a colorless oil. This oil wasanalyzed by high-performance liquid chromatography (column: DevelosilODS-HG-3 4.6×250 mm, product of Nomura Chemical, eluent:water/acetonitrile=50/50, flow rate: 1.0 mL/min, detector: UV 220 nm,column temperature: 40° C.). The compositional yield values were asfollows.

2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester: 86.0 weight %. As impurities,(5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester: 0.1 weight %;(2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: 0.1weight %;2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methylester: 5.4 weight %;2-[(4S,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester: 0.2 weight % were contained.

To the above oil was added 30 mL of methylcyclohexane, and the mixturewas cooled to −30° C. (treatment concentration: 10% (substrateweight/solution volume)). About 10 mg of seed crystals were added andthe mixture was further stirred vigorously at the same temperature for 1hour. The crystals separating out were collected by suction filtration,drained thoroughly, and washed with 10 mL of cold methylcyclohexane. Thecrystal crop was then dried in vacuo (ca 1 to 5 mmHg, 20 to 40° C., 2hours) to obtain 2.24 g of2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester as crystals (crystallization recovery rate 74%).Analysis of the above crystals showed a purity of 97.2 weight % (96.8area %). As impurities, the crystal crop contained:2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methylester: 2.8 weight % (2.8 area %);(5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester: undetected;(2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran:undetected;2-[(4S,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester: undetected.

EXAMPLE 282-[4R,6S]-6-(hydroxymethyl)-2,2-dimethyl-1,3-dioxan-4-yl]acetictert-butyl ester

To a solution of 3.64 g (10 mmol) of the2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetictert-butyl ester produced in Example 26 in methanol (36 mL) was added 10mL of 1N-aqueous sodium hydroxide solution, and the mixture was stirredat room temperature for 2 hours. This reaction mixture was adjusted topH 7 by gradual addition of 1N-hydrochoric acid under ice-cooling. Themethanol was then distilled off under reduced pressure and the residualaqueous solution was extracted with 2 portions of methylene chloride, 70mL each. The organic layer was dehydrated over anhydrous sodium sulfateand the solvent was distilled off under reduced pressure to give acolorless oil. This residue was purified by silica gel columnchromatography (Kieselgel 60, product of Merck; hexane: acetone=5:1) togive 2.34 g of2-[(4R,6S)-6-(hydroxymethyl)-2,2-dimethyl-1,3-dioxan-4-yl]acetictert-butyl ester (white solid). Yield 90%.

¹H-NMR (CDCl₃, 400 MHz/ppm); 1.29-1.52 (2H, m), 1.39 (3H, m), 1.45 (9H,s), 1.47 (3H, s), 2.05 (1H, bs), 2.33 (1H, dd), 2.44 (1H, dd), 3.47-3.53(1H, m), 3.99-4.04 (1H, m), 4.27-4.33 (1H, m)

IR (neat); 2980, 1720, 1365, 1200, 1150, 1020 cm⁻¹

INDUSTRIAL APPLICABILITY

In accordance with the present invention constituted as above,pharmaceutical intermediates, particularly optically active2-[6-(hydroxymethyl)-1,3-dioxan-4-yl]acetic acid derivatives which areof value as intermediates of HMG-COA reductase inhibitors, can beproduced from inexpensive, readily available starting materials withoutusing any extraordinary equipment such as a low-temperature reactor.

1. An isolation/purification process which comprises treating a compoundcontaminated with an impurity and represented by the following formula(V);

in the formula, R¹ represents a hydrogen, an alkyl group of 1 to 12carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl groupof 7 to 12 carbon atoms; and R⁴ represents a hydrogen, an alkyl group of1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or anaralkyl group of 7 to 12 carbon atoms, with an aliphatic hydrocarbonsolvent to remove the impurity contaminating the compound represented bythe above formula (V) and obtaining the compound represented by theabove formula (V) in a crystal form.
 2. The isolation/purificationprocess according to claim 1 wherein the impurity contaminating thecompound of the above formula (V) is a compound represented by thefollowing formula (XII);

in the formula, R¹ and R⁴ are as defined above.
 3. Theisolation/purification process according to claim 1 wherein thealiphatic hydrocarbon solvent is pentane, hexane, methylcyclohexane,heptane, octane, or isooctane.
 4. The isolation/purification processaccording to claim 1 wherein the crystallization is carried out withadditional use of an auxiliary solvent, said solvent being used for apurpose of improving at least one of a solubility, yield, treatmentconcentration, effect of purification, and physical properties ofobtainable crystals of the compound represented by the above formula(V).
 5. The isolation/purification process according to claim 4 whereinthe auxiliary solvent is used in such an amount that the weight ratio ofsaid auxiliary solvent and the aliphatic hydrocarbon solvent (saidauxiliary solvent/aliphatic hydrocarbon solvent) is not greater than 1at completion of the procedure for crystallization.
 6. Theisolation/purification process according to claim 4 wherein theauxiliary solvent is at least one species selected from the groupconsisting of toluene, ethyl acetate, methyl tert-butyl ether andmethylene chloride.
 7. The isolation/purification process according toclaim 1 wherein the compound represented by the above formula (V) isused, said compound being produced by treating a compound represented bythe following formula (IV);

in the formula, R¹ is as defined above, with an acylating agent in thepresence of a base.
 8. The isolation/purification process according toclaim 7 wherein a compound represented by the following formula (XI);

or a compound represented by the following formula (XVI);

in the above formulas, R⁴ represents a hydrogen, an alkyl group of 1 to12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkylgroup of 7 to 12 carbon atoms; and Q represents a leaving group, is usedas the acylating agent.
 9. The isolation/purification process accordingto claim 8 wherein Q of the acylating agent (XI) is a halogen atom. 10.The isolation/purification process according to claim 9 wherein thehalogen atom is a chlorine atom.
 11. The isolation/purification processaccording to claims 7 wherein an amine is used as the base.
 12. Theisolation/purification process according to claim 11 whereintriethylamine or pyridine is used as the amine used in the acylationstep.
 13. The isolation/purification process according to claim 7wherein the compound represented by the above formula (IV) is used, saidcompound being produced by reacting an enolate prepared by permitting abase or a 0-valent metal to act on an acetic acid ester derivativerepresented by the following formula (II);X¹CH₂CO₂R¹  (II) in the formula, R¹ is an defined above; and X¹represents a hydrogen or a halogen atom, with(S)-β-hydroxy-γ-butyrolactone represented by the formula (III);

at a temperature not lower than −30° C.
 14. The isolation/purificationprocess according to claim 13 wherein X¹ of the acetic acid esterderivative (II) is a hydrogen atom and a magnesium amide represented bythe following formula (VIII);

in the formula, R⁵ and R⁶ each independently represents an alkyl groupof 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, anaralkyl group of 7 to 12 carbon atoms, or a silyl group; and X²represents a halogen atom, is used as the base in preparing the enolate.15. The isolation/purification process according to claim 14 wherein, ofthe magnesium amide (VIII), each of R⁵ and R⁶ is an isopropyl group andX² is a chlorine atom.
 16. The isolation/purification process accordingto claim 13 wherein X¹ of the acetic acid ester derivative (II) is ahalogen atom and magnesium or zinc is used as the 0-valent metal inpreparing the enolate.
 17. The isolation/purification process accordingto claim 13 wherein the reaction of the enolate with(S)-β-hydroxy-γ-butyrolactone (III) is carried out in the presence of apolyether.
 18. The isolation/purification process according to claim 17wherein dimethoxyethane is used as the polyether.
 19. Theisolation/purification process according to claim 17 wherein thecompound represented by the above formula (IV) is used, said compoundbeing produced by treating, in advance, (S)-β-hydroxy-γ-butyrolactone(III) with a Grignard reagent represented by the following formula (IX);R⁷—Mg—X³  (IX) in the formula, R⁷ represents an alkyl group of 1 to 12carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl groupof 7 to 12 carbon atoms; and X³ represents a halogen atom, and reacting,at a temperature not lower than −30° C., with an enolate prepared bypermitting a base or a 0-valent metal to act on an acetic acid esterderivative (II).
 20. The isolation/purification process according toclaim 19 wherein, of the Grignard reagent (IX), R⁷ is a tert-butyl groupand X³ is a chlorine atom.
 21. The isolation/purification processaccording to claim 7 wherein the compound represented by the aboveformula (IV) is used, said compound being produced by treating, inadvance, (S)-β-hydroxy-γ-butyrolactone (III) with a base and a magnesiumcompound, and reacting, at a temperature not lower than −30° C., with anenolate prepared by permitting a base or a 0-valent metal to act on anacetic acid ester derivative (II).
 22. The isolation/purificationprocess according to claim 21 wherein the base is sodium hydride,lithium diisopropylamide, or chloromagnesium diisopropylamide.
 23. Theisolation/purification process according to claim 21 wherein themagnesium compound is magnesium chloride or magnesium bromide.
 24. Theisolation/purification process according to claim 19 wherein X¹ of theacetic acid ester derivative (II) is a hydrogen atom and a lithium amiderepresented by the following formula (X);

in the formula, R⁸ and R⁹ each independently represents an alkyl groupof 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, anaralkyl group of 7 to 12 carbon atoms, or a silyl group, is used as thebase in preparing the enolate.
 25. The isolation/purification processaccording to claim 24 wherein, of the lithium amide (X), each of R⁸ andR⁹ represents an isopropyl group.
 26. The isolation/purification processaccording to claim 19 wherein X¹ of the acetic acid ester derivative(II) is a halogen atom and magnesium or zinc is used as the 0-valentmetal in preparing the enolate.
 27. The isolation/purification processaccording to claim 1 wherein R¹ is a tert-butyl group.
 28. Theisolation/purification process according to claim 1 to wherein R⁴ is aphenyl group.
 29. A production process of a compound represented by thefollowing formula (VI);

in the formula, R¹ represents a hydrogen, an alkyl group of 1 to 12carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl groupof 7 to 12 carbon atoms; and R⁴ represents a hydrogen, an alkyl group of1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or anaralkyl group of 7 to 12 carbon atoms, which comprises reducing acompound represented by the following formula (V);

in the formula, R¹ and R⁴ are as defined above, with a microorganism,wherein the microorganism is selected from among genera and species ofmicroorganisms from the group consisting of Ashbya, Botryoascus,Brettanomyces, Citeromyces, Clavispora, Cryptococcus, Dekkera,Dipodascus, Galactomyces, Geotrichum, Hanseniaspora, Hormoascus,Hyphopichia, Issatchenkia, Kluyveromyces, Komagataella, Lipomyces,Metschnikowia, Nakazawaea, Ogataea, Pachysolen, Pichia, Rhodotorula,Rhodsporidium, Saccharomyces, Saccharomycodes, Saccharomycopsis,Saturnospora, Schizoblastosporion, Schizosaccharomyces, Schwanniomyces,Sporidiobolus, Sporobolomyces, Torulaspora, Torulopsis, Trichosporon,Willopsis, Yamadazyma, Zygosaccharomyces, Acidiphilium, Aerobacter,Alcaligenes, Arthrobacter, Aureobacterium, Bacillus, Brevibacterium,Buttiauxella, Cedecea, Cellulomonas, Citrobacter, Clostridium,Comamonas, Corynebacterium, Enterobacter, Erwinia, Escherichia,Flavobacterium, Klebsiella, Luteococcus, Microbacterium, Micrococcus,Ochrobactrum, Proteus, Providencia, Pseudomonas, Rhodococcus, Sarcina,Serratia, Sphingobacterium, Tsukamurella, Absidia, Acremonium, Aegerita,Agrocybe, Amylostereum, Aspergillus, Byssochlamys, Chaetomidium,Chaetosartorya, Cladosporium, Coprinus, Crinipellis, Endophragmia,Flavolus, Fomitopsis, Fusarium, Ganoderma, Glomerella, Laetiporus,Lentinus, Lenzites, Macrophoma, Monascus, Mortierella, Paecilomyces,Penicillium, Phialophora, Pholiota, Pleurotus, Scopulariopsis,Sehizophyllum, Sporotrichum, Zygorhynchus, Microtetraspora, andStreptomyces.
 30. The production process according to claim 29 wherein aculture broth, cells or processed cells of the microorganism is used.31. The production process according to claim 29 wherein themicroorganism is selected from the group consisting of Ashbya gossypii,Botryoascus synnaedendrus, Brettanomyces custersianus, Citeromycesmatritensis, Clavispora lusitaniae, Cryptococcus laurentii, Dekkeraanomala, Dipodascus armillariae, Dipodascus ovetensis, Dipodascustetrasperma, Galactomyces reessii, Geotrichum candidum, Geotrichumfermentans, Geotrichum fragrans, Geotrichum loubieri, Hanseniasporaguilliermondii, Hormoascus philentomus, Hormoascus platypodis,Hyphopichia burtonii, Issatchenkia orientalis, Issatchenkia terricola,Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces polysporus,Kluyveromyces thermotolerans, Komagataella pastoris, Lipomyces starkeyi,Metschnikowia bicuspidata, Metschnikowia pulcherrima, Nakazawaeaholstii, Ogataea minuta var. minuta, Ogataea pini, Ogataea polymorpha,Ogataea wickerhamii, Pachysolen tannophilus, Pichia canadensis, Pichiafarinose, Pichia jandinii, Pichia saitoi, Pichia toletana, Pichiatriangularis, Pichia wickerhamii, Rhodotorula graminis, Rhodotorulaminuta, Rhodsporidium diobovatum, Rhodsporidium toruloides,Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces rosei,Saccharomyces sake, Saccharomyces steineri, Saccaromyces unisporus,Saccharomycodes ludwigii, Saccharomycopsis capsularis, Saccharomycopsismalanga, Saturnospora dispora, Schizoblastosporion kobayasii,Schizosaccharomyces pombe, Schwanniomyces occidentalis var.occidentalis, Sporidiobolus johnsonii, Sporobolomyces pararoseus,Sporobolomyces salmonicolor, Torulaspora delbrueckii, Torulopsismethanolevescens, Torulopsis osboenis, Torulopsis sp., Torulopsis uvae,Trichosporon pullulans, Trichosporon sp. Willopsis saturnus var. mrakii,Willopsis saturnus var. saturnus, Yamadazyma farinosa, Yamadazymahaplophila, Zygosaccharomyces naniwensis, Zygosaccharomyces sp.,Acidiphilium cryptum, Aerobacter cloacae, Alcaligenes xylosoxidans,Alcaligenes xylosoxidans subsp. denitrificans, Arthrobacter globiformis,Arthrobacter protophormiae, Aureobacterium esteraromaticum, Bacillusbadius, Bacillus sphaericus, Brevibacterium ammomiagenes, Buttiauxellaagrestis, Cedecea davisiae, Cellulomonas sp., Cellulomonas turbata,Citrobacter freundii, Clostridium cylindrosporum, Comamonastestosteroni, Corynebacterium acectoacidophilum, Corynebacteriumammoniagenes, Corynebacterium glutamicum, Corynebacterium glutamicus,Enterobacter aerogenes, Enterobacter cloacae, Erwinia carotovora subsp.carotovora, Escherichia coli, Flavobacterium flavesceus, Klebsiellaplanticola, Luteococcus japonicus, Microbacterium arborescens,Micrococcus flavus, Micrococcus luteus, Ochrobactrum sp., Proteusinconstans, Proteus mirabilis, Proteus rettgeri, Proteus vulgaris,Providencia stuartii, Pseudomonas aeruginosa, Pseudomonas putida,Pseudomonas stutzeri, Rhodococcus equi, Sarcina lutea, Serratiaplymuthicum, Serratia proteamaculans subsp. proteamaculans,Sphingobacterium spiritivorum, Tsukamurella paurometabolum, Absidiaorchidis, Acremonium bacillisporum, Aegerita candida, Agrocybecylindracea, Amylostereum areolatum, Aspergillus parasiticus,Aspergillus phoenicis, Byssochlamys fulva, Chaetomidium fimeti,Chaetosartorya stromatoides, Cladosporium resinae F. avellaneum,Coprinus cinereus, Coprinus lagopus, Coprinus sp., Crinipellisstipitaria, Endophragmia alternata, Flavolus arcularius, Fomitopsispubertatis, Fusarium merismoides, Ganoderma lucidum, Glomerellacingulata, Laetiporus sulphureus, Lentinus lepideus, Lenzites betulina,Macrophoma commelinae, Monascus purpureus, Mortierella isabellina,Paecilomyces varioti, Penicillium chermesinum, Penicillium chrysogenum,Penicillium expansum, Penicillium lilacinium, Phialophora fastigiata,Pholiota aurivella, Pholiota limonella, Pleurotus dryinus, Pleurotusostreatus, Pleurotus porrigens, Scopulariopsis brevicaulis,Sehizophyllum commune, Sporotrichum aurantiacum, Zygorhynchus moelleri,Microtetraspora roseoviolacea, Streptomyces achromogenes subsp.rubradiris, Streptomyces sp. and Streptomyces aureus.
 32. The productionprocess according to claim 29 wherein R¹ is a tert-butyl group.
 33. Theproduction process according to claim 29 wherein R⁴ is a phenyl group.34. A production process of a compound represented by the followingformula (VII);

in the formula, R¹ represents a hydrogen, an alkyl group of 1 to 12carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl groupof 7 to 12 carbon atoms; R⁴ represents a hydrogen, an alkyl group of 1to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkylgroup of 7 to 12 carbon atoms; R² and R³ each independently represents ahydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; and R² andR³ may jointly form a ring, which comprises treating a compoundrepresented by the following formula (VI);

in the formula, R¹ and R⁴ are as defined above, with an acetal-formingreagent using an amine salt composed of an acid and an amine as acatalyst.
 35. The production process according to claim 34 wherein theamine salt is prepared and used in situ.
 36. The production processaccording to claim 34 wherein the acid is hydrogen chloride, hydrogenbromide, sulfuric acid, methanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid or trifluoroacetic acid.
 37. The productionprocess according to claim 34 wherein the amine is a tertiary amine. 38.The production process according to claim 37 wherein the tertiary amineis triethylamine, N-methylmorpholine, diisopropylethylamine, pyridine,2-methylpyridine, 3-methylpyridine, or imidazole.
 39. The productionprocess according to claim 34 wherein the amine is used in an excessamount relative to the acid.
 40. The production process according toclaims 34 wherein the acetal-forming reagent is 2,2-dimethoxypropane.41. The production process according to claim 34 wherein R¹ is atert-butyl group.
 42. The production process according to claim 34wherein R⁴ is a phenyl group.
 43. The production process according toclaim 34 wherein each of R² and R³ is a methyl group.
 44. Anisolation/purification process which comprises treating a compoundrepresented by the following formula (VI);

in the formula, R¹ represents a hydrogen, an alkyl group of 1 to 12carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl groupof 7 to 12 carbon atoms; and R⁴ represents a hydrogen, an alkyl group of1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or anaralkyl group of 7 to 12 carbon atoms, with an acetal-forming reagent inthe presence of an acid catalyst to thereby convert the same to acompound represented by the following formula (VII);

in the formula, R¹ and R⁴ are as defined above; R² and R³ eachindependently represents a hydrogen, an alkyl group of 1 to 12 carbonatoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7to 12 carbon atoms; and R² and R³ may jointly form a ring, treating thecompound contaminated with an impurity and represented by the aboveformula (VII) with an aliphatic hydrocarbon solvent to remove theimpurity contaminating the compound represented by the above formula(VII) and obtaining the compound represented by the above formula (VII)in a crystal form.
 45. The isolation/purification process according toclaim 44 wherein the impurity contaminating the compound represented bythe above formula (VII) is at least one compound selected from the groupconsisting of a compound represented by the following formula (XIII);

in the formula, R², R³ and R⁴ are as defined above; and R¹⁰ represents alower alkyl group and is different from R¹, a diastereomer representedby the following formula (XIV);

in the formula, R¹, R², R³, and R⁴ are as defined above, a compoundrepresented by the following formula (XV);

in the formula, R⁴ is as defined above, and a compound represented bythe following formula (VI);

in the formula, R¹ and R⁴ are as defined above.
 46. Theisolation/purification process according to claim 44 wherein thealiphatic hydrocarbon solvent is pentane, hexane, methylcyclohexane,heptane, octane, or isooctane.
 47. The isolation/purification processaccording to claim 44 wherein the crystallization is carried out withadditional use of an auxiliary solvent, said solvent being used for apurpose of improving at least one of a solubility, yield, treatmentconcentration, effect of purification, and physical properties ofobtainable crystals of the compound represented by the above formula(VII).
 48. The isolation/purification process according to claim 47wherein the auxiliary solvent is used in such an amount that the weightratio of said auxiliary solvent and the aliphatic hydrocarbon solvent(said auxiliary solvent/aliphatic hydrocarbon solvent) is not greaterthan 1 at completion of the procedure for crystallization.
 49. Theisolation/purification process according to claim 47 wherein theauxiliary solvent is at least one species selected from the groupconsisting of toluene, ethyl acetate, methyl tert-butyl ether andmethylene chloride.
 50. The isolation/purification process according toclaim 44 wherein an amine salt composed of an acid and an amine is usedas the acid catalyst.
 51. The isolation/purification process accordingto claim 50 wherein the amine salt is prepared and used in situ.
 52. Theisolation/purification process according to claim 50 wherein the acid ishydrogen chloride, hydrogen bromide, sulfuric acid, methanesulfonicacid, benzenesulfonic acid, p-toluenesulfonic acid, or trifluoroaceticacid.
 53. The isolation/purification process according to claim 50wherein the amine is a tertiary amine.
 54. The isolation/purificationprocess according to claim 53 wherein the tertiary amine istriethylamine, N-methylmorpholine, diisopropylethylamine, pyridine,2-methylpyridine, 3-methylpyridine, or imidazole.
 55. Theisolation/purification process according to claim 50 wherein the amineis used in an excess amount relative to the acid.
 56. Theisolation/purification process according to claim 44 wherein theacetal-forming reagent is 2,2-dimethoxypropane.
 57. Theisolation/purification process according to claim 44 wherein R¹ is atert-butyl group and R¹⁰ is a methyl group.
 58. Theisolation/purification process according to claim 44 wherein R⁴ is aphenyl group.
 59. The isolation/purification process according to claim44 wherein each of R and R³ is a methyl group.